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Monitoring zoonoses in urban environments is of great relevance, where the incidence of certain pathogens may be higher and where population density makes the spread of any contagious disease more likely. In this study we applied a metabarcoding approach to study potentially zoonotic pathogens in faecal samples of 9 urban vertebrate species. We applied this methodology with two objectives. Firstly, to obtain information on potential pathogens present in the urban fauna of a large European city (Madrid, Spain) and to determine which are their main reservoirs. In addition, we tested for differences in the prevalence of these potential pathogens between urban and rural European rabbits, used as ubiquitous species. Additionally, based on the results obtained, we evaluated the effectiveness of metabarcoding as a tool for monitoring potential pathogen. Our results revealed the presence of potentially zoonotic bacterial genera in all studied host species, 10 of these genera with zoonotic species of mandatory monitoring in the European Union. Based on these results, urban birds (especially house sparrows and pigeons) and bats are the species posing the greatest potential risk, with Campylobacter and Listeria genera in birds and of Chlamydia and Vibrio cholerae in bats as most relevant pathogens. This information highlights the risk associated with fresh faeces from urban wildlife. In addition, we detected Campylobacter in >50 % of the urban rabbit samples, while we only detected it in 11 % of the rural rabbit samples. We found that urban rabbits have a higher prevalence of some pathogens relative to rural rabbits, which could indicate increased risk of pathogen transmission to humans. Finally, our results showed that metabarcoding can be an useful tool to quickly obtain a first screening of potentially zoonotic organisms, necessary information to target the monitoring efforts on the most relevant pathogens and host species., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Juan Traba reports financial support was provided by Community of Madrid. Juan Traba reports article publishing charges was provided by Community of Madrid. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

The Western Mediterranean coast is under the influence of anthropogenic pressures, including land use, increasing amounts of dangerous waste and habitat destruction. In 2021, the French RINBIO network (http://www.ifremer.fr/envlit/) originally dedicated to assess chemical contamination in the region, focused on biological effects produced by contaminants and the interaction with natural variability in mussels using an active caging strategy. Cell and tissue level biomarkers were applied for 17 sampling sites divided in three sub-regions categorized by different environmental conditions. Results provide critical information for ecosystem health assessment using mussels as sentinel species in the Western Mediterranean Sea. The influence of natural and confounding factors (trophic condition, reproductive cycle, caging strategy), on biological responses to mild chemical contamination, was discussed and discriminated for health status assessment. Results provide valuable data available as reference values for the assessment of biomarkers and histopathological alterations for large-scale active biomonitoring campaigns in the Western Mediterranean Sea., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Mammal species globally exhibit distribution patterns conditioned by environmental conditions and human impact. The Mediterranean basin provides an ideal system to study these effects due to its diverse climate, and habitat conditions. In this work, we aim to assess the impact of landscape heterogeneity and anthropization degree on terrestrial mammal diversity in this region. Accordingly, we deployed over 300 camera traps across 28 sites for 3 months. Detected mammal species (weighing more than 1kg) were classified as domestic carnivores, domestic ungulates, wild carnivores, wild ungulates, lagomorphs, and large rodents. Alpha and beta diversity were calculated for each group and all wild mammals. Simple linear regressions and multimodal analysis were conducted between mammal diversities and climate, environmental conditions, landscape heterogeneity, and anthropization degree variables. Redundancy analyses were performed to identify variables and species determining the mammalian community composition. Indexes measuring landscape heterogeneity, anthropization degree, and its 30-year change did not correlate with mammal diversity. However, the difference in elevation within sites and domestic carnivore abundance showed a significant positive correlation with some of the diversity indexes. Nonetheless, rainfall and mean elevation factors generally showed the highest correlation with mammal diversity. Instead, a few influential species, including generalists and open-habitat specialists, highlighted the importance of conserving open areas, as well as the importance of the Pyrenees region as a key habitat for certain species. Therefore, climatic variables emerged as the key determinants of mammal diversity, highlighting climate change as a potential threat to mammal diversity in this area., (© 2024 The Author(s). Integrative Zoology published by International Society of Zoological Sciences, Institute of Zoology/Chinese Academy of Sciences and John Wiley & Sons Australia, Ltd.)

The study of trophic niche partitioning is of great importance for understanding community structure and species coexistence, particularly if these are threatened. Here DNA metabarcoding was used to assess the diet of four threatened steppe bird species (two bustards and two sandgrouses), with the aim of better understanding their dietary requirements, trophic interactions, and potential threats. The results showed seasonal and interspecific differences in their plant diet, with greater importance of cultivated plants during autumn and winter (around 50 % of their diet) than spring. Plants of the genus Convolvulus and of the family Brassicaceae were frequently consumed by all species. In spring, poppies (Papaver spp.) were a considerable part of their diet, and could be used as a source of carotenoids or for their anti-parasitic properties. Furthermore, results evidenced a trophic niche partitioning among species, with a marked segregation between bustard species and, to a lesser extent, between sandgrouse species. Diet similarity was generally higher between species from different orders that occur in mixed-species flocks (bustard - sandgrouse) than between species of the same order. This partitioning was probably related to a stratification in habitat use rather than to specialisation and might prevent competition to some extent. However, the homogenization of trophic resources resulting from agricultural intensification could pose an important threat, particularly during autumn, when weeds are scarcer and the most abundant trophic resource are sown seeds, which are often treated with pesticides., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Soil contamination by antibiotics is a global issue of great concern that contributes to the rise of bacterial antibiotic resistance and can have toxic effects on non-target organisms. This study evaluated the variations of molecular, cellular, and histological parameters in Eisenia fetida earthworms exposed to sulfamethazine (SMZ) and tetracycline (TC), two antibiotics commonly found in agricultural soils. The earthworms were exposed for 14 days to a series of concentrations (0, 10, 100, and 1000 mg/kg) of both antibiotics. SMZ and TC did not affect the survival of E. fetida, however, other effects at different levels of biological complexity were detected. The two highest concentrations of SMZ reduced the viability of coelomocytes. At the highest TC concentration, there was a noticeable decline in cell viability, acetylcholinesterase activity (neurotoxicity), and the relative presence of mucopolysaccharides in the epidermis (mucous production). Glutathione S-transferase activity decreased in all TC treatments and at the highest SMZ concentration. However, levels of malondialdehyde and protein carbonyls did not change, suggesting an absence of oxidative stress. Tetracycline was neurotoxic to E. fetida and changed the integrity of the epidermis. Both antibiotics altered the intestinal microbiota of E. fetida, leading to a reduction in the relative abundance of bacteria from the phyla Proteobacteria and Bacteroidetes, while causing an increase in the phylum Actinobacteroidota. All observed changes indicate that both SMZ and TC can disrupt the earthworms' immune system and gut microbiome, while fostering the growth of bacteria that harbour antibiotic resistance genes. Finally, both antibiotics exerted additional metabolic and physiological effects that increased the vulnerability of E. fetida to pathogens., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Marine and freshwater mammalian predators and fish samples, retrieved from environmental specimen banks (ESBs), natural history museum (NHMs) and other scientific collections, were analysed by LIFE APEX partners for a wide range of legacy and emerging contaminants (2545 in total). Network analysis was used to visualize the chemical occurrence data and reveal the predominant chemical mixtures for the freshwater and marine environments. For this purpose, a web tool was created to explore these chemical mixtures in predator-prey pairs. Predominant chemicals, defined as the most prevalent substances detected in prey-predator pairs were identified through this innovative approach. The analysis established the most frequently co-occurring substances in chemical mixtures from AP&P in the marine and freshwater environments. Freshwater and marine environments shared 23 chemicals among their top 25 predominant chemicals. Legacy chemical, including perfluorooctanesulfonic acid (PFOS), brominated diphenyl ethers (BDEs), polychlorinated biphenyls (PCBs), hexachlorobenzene and mercury were dominant chemicals in both environments. Furthermore, N-acetylaminoantipyrine was a predominant pharmaceutical in both environments. The LIFE APEX chemical mixture application (https://norman-data.eu/LIFE_APEX_Mixtures) was proven to be useful to establish most prevalent compounds in terms of number of detected counts in prey-predator pairs. Nonetheless, further research is needed to establish food chain associations of the predominant chemicals., Competing Interests: Declaration of competing interest All the authors declare no conflict of interest., (Copyright © 2024. Published by Elsevier B.V.)

Biological effects of aqueous fractions of a crude oil, alone or in combination with dispersant, were investigated in mussels, Mytilus edulis, exposed at three temperatures (5, 10 and 15 °C). Polycyclic aromatic hydrocarbons (PAHs) tissue concentrations were determined, together with genotoxicity, oxidative stress and general stress biomarkers and the Integrated Biological Response (IBR) index. The bioaccumulation of individual PAHs varied depending on the exposure temperature, with relevant bioaccumulation of phenantrene and fluoranthene at 5 °C and heavier (e.g. 5-rings) PAHs at 15 °C. The values and response profiles of each particular biomarker varied with exposure time, concentration of the oil aqueous fraction and dispersant addition, as well as with exposure temperature. Indeed, PAH bioaccumulation and biomarker responsiveness exhibited specific recognizable patterns in mussels exposed at low temperatures. Thus, genotoxicity was recorded early and transient at 5 °C and delayed but unremitting at 10-15 °C. Catalase activity presented a temperature-dependent response profile similar to the genotoxicity biomarker; however, glutathione-S-transferase responsiveness was more intricate. Lysosomal membrane stability in digestive cells decreased more markedly at 5 °C than at higher temperatures and the histological appearance of the digestive gland tissue was temperature-specific, which was interpreted as the combined effects of PAH toxicity and cold stress. It can be concluded that the profile and level of the biological effects are definitely different at low temperatures naturally occurring in the Arctic/Subarctic region (e.g. 5 °C) than at higher temperatures closer to the thermal optimum of this species (10-15 °C)., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

The presence of nanoplastics (NPs) and microplastics (MPs) in the environment is recognised as a global-scale problem. Due to their hydrophobic nature and large specific surface, NPs and MPs can adsorb other contaminants, as polycyclic aromatic hydrocarbons (PAHs), and modulate their bioavailability and hazard. Adult zebrafish were exposed for 3 and 21 days to: (1) 0.07 mg/L NPs (50 nm), (2) 0.05 mg/L MPs (4.5 μm), (3) MPs with sorbed oil compounds of the water accommodated fraction (WAF) of a naphthenic crude oil (MPs-WAF), (4) MPs with sorbed benzo(a)pyrene (MPs-B(a)P), (5) 5 % WAF and (6) 21 μg/L B(a)P. Electrodense particles resembling NPs were seen in the intestine lumen close to microvilli. MPs were abundantly found in the intestine lumen, but not internalised into the tissues. After 21 days, NPs caused a significant downregulation of cat, and upregulation of gpx1a and sod1, while MPs upregulated cyp1a and increased the prevalence of liver vacuolisation. No histopathological alteration was observed in gills. In this study, contaminated MPs did not increase PAH levels in zebrafish but results highlight the potential differential impact of plastic particles depending on their size, making it necessary to urgently address the ecotoxicological impact of real environmental NPs and MPs., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

The delay in making a correct diagnosis of Candida auris causes concern in the healthcare system setting, and immunoproteomics studies are important to identify immunoreactive proteins for new diagnostic strategies. In this study, immunocompetent murine systemic infections caused by non-aggregative and aggregative phenotypes of C. auris and by Candida albicans and Candida haemulonii were carried out, and the obtained sera were used to study their immunoreactivity against C. auris proteins. The results showed higher virulence, in terms of infection signs, weight loss, and histopathological damage, of the non-aggregative isolate. Moreover, C. auris was less virulent than C. albicans but more than C. haemulonii . Regarding the immunoproteomics study, 13 spots recognized by sera from mice infected with both C. auris phenotypes and analyzed by mass spectrometry corresponded to enolase, phosphoglycerate kinase, glyceraldehyde-3-phosphate dehydrogenase, and phosphoglycerate mutase. These four proteins were also recognized by sera obtained from human patients with disseminated C. auris infection but not by sera obtained from mice infected with C. albicans or Aspergillus fumigatus . Spot identification data are available via ProteomeXchange with the identifier PXD049077. In conclusion, this study showed that the identified proteins could be potential candidates to be studied as new diagnostic or even therapeutic targets for C. auris .

Although the 16S rRNA gene is frequently used as a phylogenetic marker in analysis of environmental DNA, this marker often fails to distinguish closely related species, including those in the genus Vibrio. Here, we investigate whether inclusion and analysis of 23S rRNA sequence can help overcome the intrinsic weaknesses of 16S rRNA analyses for the differentiation of Vibrio species. We construct a maximum likelihood 16S rRNA gene tree to assess the use of this gene to identify clades of Vibrio species. Within the 16S rRNA tree, we identify the putative informative bases responsible for polyphyly, and demonstrate the association of these positions with tree topology. We demonstrate that concatenation of 16S and 23S rRNA genes increases the number of informative nucleotide positions, thereby overcoming ambiguities in 16S rRNA-based phylogenetic reconstructions. Finally, we experimentally demonstrate that this approach considerably improves the differentiation and identification of Vibrio species in environmental samples., (© 2024. The Author(s).)

In this work, an accurate analytical method was developed for the simultaneous analysis of twenty-seven antimicrobials (AMs) in earthworms using liquid chromatography coupled to a triple quadrupole mass spectrometry detector (UHPLC-MS/MS). Adequate apparent recoveries (80-120 %) and limits of quantification (LOQ) (1 μg·kg -1 - 10 μg·kg -1 ) were obtained, with the exception of norfloxacin (34 μg·kg -1 ). The method was applied to evaluate the accumulation of sulfamethazine (SMZ) and tetracycline (TC) in earthworms after performing OECD-207 toxicity test, in which Eisenia fetida (E. fetida) organisms were exposed to soils spiked with 10 mg·kg -1 , 100 mg·kg -1 or 1000 mg·kg -1 of SMZ and TC, individually. The results confirmed the bioaccumulation of both AMs in the organisms, showing a greater tendency to accumulate SMZ since higher bioconcentration factor values were obtained for this compound at the exposure concentrations tested. In addition, the degradation of both AMs in both matrices, soils and earthworms was studied using liquid chromatography coupled to a q-Orbitrap high resolution mass spectrometry detector. Thirteen transformation products (TPs) were successfully identified, eight of them being identified for the first time in soil/earthworm (such as 4-Amino-3-chloro-n-(4,6-dimethylpyrimidin-2-yl)benzenesulfonamide or 4-(dimethylamino)-1,11,12a-trihydroxy-6,6-dimethyl-3,7,10,12-tetraoxo-3,4,4a,5,5a,6,7,10,12,12a-decahydrotetracene-2-carboxamide, among others) and their formation/degradation trend over time was also studied. Regarding the biological effects, only SMZ caused changes in earthworm growth, evidenced by weight loss in earthworms exposed to concentrations of 100 mg·kg -1 and 1000 mg·kg -1 . Riboflavin decreased at all concentrations of SMZ, as well as at the highest concentration of TC. This indicates that these antibiotics can potentially alter the immune system of E. fetida. This research represents a significant advance in improving our knowledge about the contamination of soil by AM over time. It investigates the various ways in which earthworms are exposed to AMs, either by skin contact or ingestion. Furthermore, it explores how these substances accumulate in earthworms, the processes by which earthworms break them down or metabolise them, as well as the resulting TPs. Finally, it examines the potential effects of these substances on the environment., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

A toxicogenomic approach was used for toxicity evaluation of arsenic in the aquatic environment, and differential gene expression was investigated from 24 h and 96 h water-only acute toxicity tests with the aquatic oligochaete, Tubifex tubifex ( Annelida , Clitellata ). Several toxicological endpoints (survival and autotomy) of the oligochaete and tissue residues were measured, and dose-response modelling of gene expression data was studied. A reference transcriptome of the aquatic oligochaete, T. tubifex , was reconstructed for the first time, and genes related to cell stress response ( Hsc70 , Hsp10 , Hsp60 , and Hsp83 ), energy metabolism ( COX1 ), oxidative stress ( Cat , GSR , and MnSOD ), and the genes involved in the homeostasis of organisms ( CaM , RpS13 , and UBE2 ) were identified and characterised. The potential use of the genes identified for risk assessment in freshwater ecosystems as early biomarkers of arsenic toxicity is discussed.

Microplastics are a global ecological concern due to their potential risk to wildlife and human health. Animals ingest microplastics, which can enter the trophic chain and ultimately impact human well-being. The ingestion of microplastics can cause physical and chemical damage to the animals' digestive systems, affecting their health. To estimate the risk to ecosystems and human health, it is crucial to understand the accumulation and localization of ingested microplastics within the cells and tissues of living organisms. However, analyzing this issue is challenging due to the risk of sample contamination, given the ubiquity of microplastics. Here, an analytical approach is employed to confirm the internalization of microplastics in cryogenic cross-sections of mussel tissue. Using 3D Raman confocal microscopy in combination with chemometrics, microplastics measuring 1 μm in size were detected. The results were further validated using optical and fluorescence microscopy. The findings revealed evidence of microplastics being internalized in the digestive epithelial tissues of exposed mussels (Mytilus galloprovincialis), specifically within the digestive cells forming digestive alveoli. This study highlights the need to investigate the internalization of microplastics in organisms like mussels, as it helps us understand the potential risks they pose to aquatic biota and ultimately to human health. By employing advanced imaging techniques, challenges associated with sample contamination can be overcome and valuable insights into the impact of microplastics on marine ecosystems and human consumers are provided., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

The Spanish and Portuguese Speaking Working Group of the International Society for Forensic Genetics (GHEP-ISFG) organized a collaborative study on mutations of Y-chromosomal short tandem repeats (Y-STRs). New data from 2225 father-son duos and data from 44 previously published reports, corresponding to 25,729 duos, were collected and analyzed. Marker-specific mutation rates were estimated for 33 Y-STRs. Although highly dependent on the analyzed marker, mutations compatible with the gain or loss of a single repeat were 23.2 times more likely than those involving a greater number of repeats. Longer alleles (relatively to the modal one) showed to be nearly twice more mutable than the shorter ones. Within the subset of longer alleles, the loss of repeats showed to be nearly twice more likely than the gain. Conversely, shorter alleles showed a symmetrical trend, with repeat gains being twofold more frequent than reductions. A positive correlation between the paternal age and the mutation rate was observed, strengthening previous findings. The results of a machine learning approach, via logistic regression analyses, allowed the establishment of algebraic formulas for estimating the probability of mutation depending on paternal age and allele length for DYS389I, DYS393 and DYS627. Algebraic formulas could also be established considering only the allele length as predictor for DYS19, DYS389I, DYS389II-I, DYS390, DYS391, DYS393, DYS437, DYS439, DYS449, DYS456, DYS458, DYS460, DYS481, DYS518, DYS533, DYS576, DYS626 and DYS627 loci. For the remaining Y-STRs, a lack of statistical significance was observed, probably as a consequence of the small effective size of the subsets available, a common difficulty in the modeling of rare events as is the case of mutations. The amount of data used in the different analyses varied widely, depending on how the data were reported in the publications analyzed. This shows a regrettable waste of produced data, due to inadequate communication of the results, supporting an urgent need of publication guidelines for mutation studies., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

The recreational boating sector is a major vector for the introduction of non-indigenous species (NIS) via biofouling. Despite applying control measures to prevent the growth of fouling communities, most vessels are NIS carriers. This study assessed the effectiveness of different antifouling strategies in a manipulative experiment by testing two common coating typologies (biocide-based and foul-release coatings), accompanied with simulated maintenance practices. The experiment was carried out in the Gulf of La Spezia (Italy) and samples were collected at two different periods. Results showed significant differences among antifouling treatments regarding community structure, diversity, coverage and biovolume of the sessile component, alongside a significant decrease in the performance of biocide-based coating with time. Interestingly, peracarid NIS/native species ratio was higher for biocide-based treatments, suggesting potential biocide resistance. This study highlights the urgent need to develop common and feasible biofouling management plans and provides insights towards identification of best practices for recreational vessels., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

There is a growing concern about the presence of pharmaceuticals on the aquatic environment, while the marine environment has been much less investigated than in freshwater. Marine mammals are suitable sentinel species of the marine environment because they often feed at high trophic levels, have unique fat stores and long lifespan. Some small delphinids in particular serve as excellent sentinel species for contamination in the marine environment worldwide. To the best of our knowledge, no pharmaceuticals have been detected or reported in dolphins so far. In the present study, muscle, liver and blubber samples from three common dolphins (Delphinus delphis) and seven striped dolphins (Stenella coeruleoalba) stranded along the Basque Coast (northern Spain) were collected. A total of 95 pharmaceuticals based on detectability and predicted ability to bioaccumulate in fish were included in the liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis. At least one pharmaceutical was found in 70 % of the individuals. Only three of the 95 monitored pharmaceuticals were detected in dolphin's tissues. Very low concentrations (<1 ng/g) of orphenadrine and pizotifen were found in liver and promethazine in blubber. Herein, the gap in the knowledge regarding the study organisms and marine environments with respect to pharmaceutical pollution, which demands further research to understand if pharmaceuticals are a threat for these apex predators, is highlighted and discussed., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

Fish display diverse reproductive strategies and their gametogenesis is influenced by numerous genetic, physiological and environmental factors. The analysis of 5S rRNA expression levels in gonads has been proposed as useful method for the molecular identification of the presence of oocytes in fish tissues. The present method provides an easy and unbiased approach to analyse the expression of tRNAs and 5S rRNA in teleost gonads and stablish the presence and developmental stage of oocytes. Total RNA extracted from gonads is analysed through capillary electrophoresis in a Bioanalyzer 2100 (Agilent Technologies) using Small RNA Assays. Electropherograms allow quantifying the concentrations of tRNAs, 5S rRNA and 5.8S rRNA per sample and calculate their tRNA/5.8S rRNA and 5S/5.8S rRNA indices. Both indices clearly differentiate ovaries from testes and can be used to identify testes that present oocytes due to exposure to environmental xenoestrogens. The tRNA/5.8S and 5S/5.8S indices show the highest values in ovaries in previtellogenic stage, values decreasing as they advance towards maturity.•Detailed molecular method to sex fish and quantitatively identify the maturity stage of females.•tRNA levels in gonads can help in the study of teleost reproduction (female fecundity assessment, molecular gonad sexing) and environmental health assessment., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 The Authors. Published by Elsevier B.V.)

The rise and fall of the Roman Empire was a socio-political process with enormous ramifications for human history. The Middle Danube was a crucial frontier and a crossroads for population and cultural movement. Here, we present genome-wide data from 136 Balkan individuals dated to the 1 st millennium CE. Despite extensive militarization and cultural influence, we find little ancestry contribution from peoples of Italic descent. However, we trace a large-scale influx of people of Anatolian ancestry during the Imperial period. Between ∼250 and 550 CE, we detect migrants with ancestry from Central/Northern Europe and the Steppe, confirming that "barbarian" migrations were propelled by ethnically diverse confederations. Following the end of Roman control, we detect the large-scale arrival of individuals who were genetically similar to modern Eastern European Slavic-speaking populations, who contributed 30%-60% of the ancestry of Balkan people, representing one of the largest permanent demographic changes anywhere in Europe during the Migration Period., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Regenerative abilities vary dramatically across animals. Even amongst planarian flatworms, well-known for complete regeneration from tiny body fragments, some species have restricted regeneration abilities while others are almost entirely regeneration incompetent. Here, we assemble a diverse live collection of 40 planarian species to probe the evolution of head regeneration in the group. Combining quantification of species-specific head-regeneration abilities with a comprehensive transcriptome-based phylogeny reconstruction, we show multiple independent transitions between robust whole-body regeneration and restricted regeneration in freshwater species. RNA-mediated genetic interference inhibition of canonical Wnt signalling in RNA-mediated genetic interference-sensitive species bypassed all head-regeneration defects, suggesting that the Wnt pathway is linked to the emergence of planarian regeneration defects. Our finding that Wnt signalling has multiple roles in the reproductive system of the model species Schmidtea mediterranea raises the possibility that a trade-off between egg-laying, asexual reproduction by fission/regeneration and Wnt signalling drives regenerative trait evolution. Although quantitative comparisons of Wnt signalling levels, yolk content and reproductive strategy across our species collection remained inconclusive, they revealed divergent Wnt signalling roles in the reproductive system of planarians. Altogether, our study establishes planarians as a model taxon for comparative regeneration research and presents a framework for the mechanistic evolution of regenerative abilities., (© 2023. The Author(s).)

Lead (Pb) and lithium (Li) are metals which have been detected in the environment and, at high concentrations, can induce toxic effects that disturb the growth, metabolism or reproduction of organisms along the entire trophic chain. The impacts of these metals have scarcely been investigated using marine bivalves, especially when acting as a mixture. The present study aimed to investigate the influence of temperature on the ecotoxicological effects caused by Pb and Li, acting alone and as a mixture, on the mussel species Mytilus galloprovincialis after 28 days of exposure. The impacts were evaluated under actual (17 °C) and projected (+4 °C) warming conditions, to understand the influence of temperature rise on the effects of the metals (both acting alone or as a mixture). The results obtained showed that the increased temperature did not influence the accumulation of metals. However, the biomarkers evaluated showed greater responses in mussels that are exposed to metals under increased temperature (21 °C). The IBR index showed that there is a comparable toxic effect of Li and Pb separately, while exposure to a mixture of both pollutants causes a significantly higher stress response. Overall, the results obtained revealed that temperature may cause extra stress on the mussels and exposure to the metal mixture caused the greatest impacts compared to each metal acting alone., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Recent research has confirmed the efficiency of insectivorous bats as pest suppressors, underlining the ecological services they offer in agroecosystems. Therefore, some efforts try to enhance bat foraging in agricultural landscapes by acting upon environmental factors favouring them. In this study, we monitored a Miniopterus schreibersii colony, in the southern Iberian Peninsula. We intensively sampled their faeces and analysed them by metabarcoding to describe how the bent-winged bat diet would change with time, and to test whether their most-consumed prey would seasonally depend on different landscapes or habitats. Our results confirm that M. schreibersii are selective opportunist predators of moths, dipterans, mayflies, and other fluttering insects, shifting their diet to temporary peaks of prey availability in their foraging range, including both pest and non-pest insects. Supporting our hypothesis, throughout the year, M. schreibersii consume insects linked to diverse open habitats, including wetlands, grassland, diverse croplands, and woodland. The importance of each prey habitat varies seasonally, depending on their insect phenology, making bats indirectly dependent on a diverse landscape as their primary prey source. Bats' predation upon pest insects is quantitatively high, consuming around 1610 kg in 5 months, of which 1467 kg correspond to ten species. So, their suppression effect may be relevant, mainly in patchy heterogeneous landscapes, where bats' foraging may concentrate in successive outbursts of pests, affecting different crops or woodlands. Our results stress that to take advantage of the ecosystem services of bats or other generalist insectivores, keeping the environmental conditions they require to thrive, particularly a heterogeneous landscape within the colony's foraging area, is crucial., (© 2023. The Author(s).)

North Atlantic and Arctic Oceans contain large amount of undiscovered oil and gas reserves. Therefore threat of oil spills and its hazardous ecological consequences are of great importance to the marine environment. Although mussels (Mytilus sp.) respond clearly to contaminants, biomarkers have shown variability linked to biological and environmental changes. In order to help avoiding misinterpretation of biological responses the aim of this study was to reveal the effect of natural variability in the responsiveness to pollution of a battery of cell and tissue-level biomarkers in mussels. Mussels were collected in relatively non-impacted and potentially impacted sites at ports and the vicinity of a waste water treatment plant in Trondheim and Tromsø in autumn of 2016. Although the battery of biomarkers used herein proved to be useful to discriminate impacted and non-impacted mussel populations, some confounding factors altering the biological responses were identified. Geographical/latitudinal factors seemed to be critical regarding the reproductive cycle, reserve material storage and the prevalence of parasites such as Gymnophallus cf. Bursicola trematodes. Mussels from the reference site in Tromsø displayed general stress responses at different levels, which could be influenced by the pathogenic effect of the Gymnophallus cf. Bursicola trematode and by a more advanced gametogenic developmental stage compared to the mussels from Trondheim, which could lead to misinterpretation of the reasons behind the measured stress levels in those mussels. Despite these confounding effects, the use of integrative tools such as IBR index helped to discriminate mussel populations from chemically impacted and non-impacted sites. Overall, this work serves as an anchor point both as a reference of the baseline level values of the analyzed endpoints in the studied geographical area and time of the year, and as an indication of the potential extent of the environmental confounding factors in monitoring programs causing stress on the analyzed mussel populations., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The use of Plant Protection Products (PPPs) is leading to high exposure scenarios with potential risk to soil organisms, including non-target species. Assessment of the effects of PPPs on non-target organisms is one of the most important components of environmental risk assessment (ERA) since they play crucial functions in ecosystems, being main driving forces in different soil processes. As part of the framework, EFSA is proposing the use of the ecosystem services approach for setting specific protection goals. In fact, the services provided by soil organisms can be impacted by the misuse of PPPs in agroecosystems. The aim of this work was to assess PPPs potential risk upon ecosystem services along European soils, considering impacts on earthworms and collembola. Four well-known (2 insecticides-esfenvalerate and cyclaniliprole- and 2 fungicides - picoxystrobin and fenamidone-) worst case application (highest recommended application) were studied; exploring approaches for linked observed effects with impacts on ecosystem services, accounting for their mode of action (MoA), predicted exposure, time-course effects in Eisenia fetida and Folsomia sp. and landscape variability. The selected fungicides exerted more effects than insecticides on E. fetida, whereas few effects were reported for both pesticides regarding Folsomia sp. The most impacted ecosystem services after PPP application to crops appeared to be habitat provision, soil formation and retention, nutrient cycling, biodiversity, erosion regulation, soil remediation/waste treatment and pest and disease regulation. The main factors to be taken into account for a correct PPP use management in crops are discussed., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Inc.)

Seed treatment with pesticides is an extended agricultural practice with a high risk to granivorous birds that consume those seeds. To characterize that risk, it is necessary to understand the ecological factors that determine the exposure chances of birds to treated seeds. We investigated how pesticide uptake by red-legged partridges was related to cultivated plant ingestion and to the use of recently sown fields. We analyzed pesticide residues in 144 fecal samples from 32 flocks and determined the plant diet composition using DNA metabarcoding. Habitat use was studied through the monitoring of 15 GPS-tagged partridges. We confirmed, through the analysis of seeds, that >80% of cereal fields from the area had seeds treated with triazole fungicides. Tebuconazole was detected in 16.6% of partridges' feces. During the sowing season, cultivated plants accounted for half of the plant diet, but no association was found between cultivated plant consumption and pesticide intake. GPS tracking revealed that tebuconazole was detected in feces when partridges had recently used sown fields, whereas nonexposed partridges showed no overlap with recently sown areas. Our results highlight the need to incorporate field ecology into the characterization of pesticide exposure to improve the efficacy of environmental risk assessment.

In recent field studies, suspected gymnophallid metacercariae were histologically located in the mantle of mussels from the Norwegian Sea. Mussels from the sites in which that infection was detected also presented abnormally high pearl numbers. It has been previously described that gymnophallid metacercariae could cause pearl formation processes in mussels, as a host reaction to encapsulate these metacercariae. Given the pathological host reaction these parasites elicit, a study was performed to identify gymnophallid metacercariae found in mussels collected from Tromsø at morphological and molecular level and to assess, by the use of molecular tools, the relationship between the parasite and the biological material inside the pearls. As a result, Gymnophallus bursicola metacercariae infecting Norwegian Mytilus edulis were identified according to morphological characters, along with the first 18S rDNA and COI sequences for this trematode species. In addition, parasite DNA from the core of the pearls was extracted and amplified for the first time, confirming the parasitological origin of these pearls. This procedure could allow identifying different parasitic organisms responsible for the generation of pearls in bivalves.

Few African Americans have been able to trace family lineages back to ancestors who died before the 1870 United States Census, the first in which all Black people were listed by name. We analyzed 27 individuals from Maryland's Catoctin Furnace African American Cemetery (1774-1850), identifying 41,799 genetic relatives among consenting research participants in 23andMe, Inc.'s genetic database. One of the highest concentrations of close relatives is in Maryland, suggesting that descendants of the Catoctin individuals remain in the area. We find that many of the Catoctin individuals derived African ancestry from the Wolof or Kongo groups and European ancestry from Great Britain and Ireland. This study demonstrates the power of joint analysis of historical DNA and large datasets generated through direct-to-consumer ancestry testing.

Traditionally, researchers have assessed diet selection by comparing consumed versus available taxa. However, taxonomic assignment is probably irrelevant for predators, who likely base their selection on characteristics including prey size, habitat, or behavior. Here, we use an aquatic insectivore, the threatened Pyrenean Desman ( Galemys pyrenaicus ), as a model species to assess whether biological traits help unravel the criteria driving food and habitat preferences. We reanalyzed data from a previous taxonomy-based study of prey selection in two contrasting streams, one with excellent conservation status and the other affected by diversion for hydropower and forestry. Available and consumed prey were characterized according to nine biological traits, and diet selection was estimated by comparing availability-measured from Surber net samples, and consumption-analyzed by metabarcoding desman feces. Traits offered a biologically coherent image of diet and almost identical selection patterns in both streams, depicting a highly specialized rheophilic predator. Desmans positively selected prey with a preference for fast flow and boulder substrate, indicating their preferred riffle habitat. On the other hand, they positively selected prey with larger but not the largest potential size, living in the water column or the litter, and not inside sediments. They also chose agile prey, swimmers or prey attached to the substrate, prey with high body flexibility, and prey living exposed and clustered in groups. Overall, our results offer a picture of desman diet preference and point to biological traits as being better than taxonomic identity to describe the diet preference of consumers., (© The Author(s) 2023. Published by Oxford University Press on behalf of the American Society of Mammalogists.)

Over the last decade, xenoestrogenic effects have been reported in populations of thicklip grey mullet Chelon labrosus from contaminated estuaries in the Bay of Biscay, resulting in intersex condition. To understand the level of gene flow in individuals of different Basque estuaries microsatellite markers were used to evaluate the population structure and connectivity of C. labrosus from estuaries of the Basque coast. 46 microsatellites were tested and 10 validated for the analysis of 204 individuals collected from 5 selected Basque estuaries and 2 outgroups in the Bay of Cadiz and Thermaic Gulf. The polymorphic microsatellites revealed 74 total alleles, 2-19 alleles per locus. The mean observed heterozygosity (0.49 ± 0.02) was lower than the expected one (0.53 ± 0.01). There was no evidence of genetic differentiation (F ST  = 0.0098, P = 0.0000) among individuals or sites. Bayesian clustering analysis revealed a single population in all sampled locations. The results of this study indicate widespread genetic homogeneity and panmixia of C. labrosus across the current sampling areas spanning the Atlantic and Mediterranean basins. The hypothesis of panmixia could therefore be well supported so individuals inhabiting estuaries with high prevalence of intersex condition should be considered as members of the same single genetic group as those inhabiting adjacent estuaries without incidence of xenoestrogenicity., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Oil spills pose significant environmental risks, particularly in cold seas. In the Baltic Sea, the low salinity (from 0 to 2 up to 18) affects the behaviour of the spilled oil as well as the efficiency and ecological impacts of oil spill response methods such as mechanical collection and the use of dispersants. In the present study, mussels (Mytilus trossulus) were exposed under winter conditions (5 °C) to the water-accommodated fraction (WAF) of Naphthenic North Atlantic crude oil prepared by mechanical dispersion or to the chemically enhanced fraction (CEWAF) obtained using the dispersant Finasol OSR 51 at salinities of 5.6 and 15.0. Especially at the lower salinity, high bioaccumulation of polycyclic aromatic hydrocarbons was recorded in mussels in the CEWAF treatments, accompanied by increased biomarker responses. In the WAF treatments these impacts were less evident. Thus, the use of dispersants in the Baltic Sea still needs to be carefully considered., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Intersex gonads have been observed in thicklip grey mullet Chelon labrosus, inhabiting estuaries with high burdens of xenoestrogens in the Southeast Bay of Biscay, but knowledge of population connectivity among estuaries is lacking for this euryhaline fish species. This study investigates the population structure of C. labrosus using otolith shape and elemental signatures of 60 adults (overall length ∼ 38 cm) from two estuaries 21 nautic miles apart, one with a high incidence of intersex condition (Gernika), and the other one pristine (Plentzia). Otolith shape analyses were performed using elliptical Fourier descriptors, while elemental signatures of whole sagittae were obtained by inductively coupled plasma mass spectrophotometry. Univariate and multivariate statistics were applied to determine if otolith signatures show patterns of homogeneity between estuaries. The data indicated significant differences in the otolith shape and elemental composition between mullets of Gernika and Plentzia. Elemental differences were mainly driven by Sr, Li (both higher in Plentzia) and Ba (higher in Gernika). The 98% re-classification success rate obtained from stepwise linear discriminant function analysis suggests that Gernika and Plentzia individuals form separated population units. The limited connectivity between these two close estuaries would indicate a different life history of exposure to chemicals, which might explain the high prevalence of intersex condition in Gernika and its absence in Plenztia., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper, (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The groundwater worms of the family Lumbriculidae exhibit high diversity and endemism in southern Europe. All stygobiont lumbriculid species known so far in this region belong to six genera. In the present study, a new stygobiont lumbriculid Fendia cantabrica n. gen. n. sp. is described, mainly based on distinct characteristics of the male duct structure. The male duct is semiprosoporous and the spermathecae (two pairs) are postatrial, starting in the ovarian segment. These characteristics are shared with only two lumbriculid genera: the Nearctic Eremidrilus and Holarctic Trichodrilus. The new genus is clearly distinguished from the former by the absence of proboscis, and from both genera by a remarkably complex structure of the atrium: a very strong musculature organized in several crossed layers, a protrusible penis with a singular hydrostatic skeleton, and two prostate glands, which join separately to the atrial ampulla by short stalks.

Graphene oxide (GO) has gained a great scientific and economic interest due to its unique properties. As incorporation of GO in consumer products is rising, it is expected that GO will end up in oceans. Due to its high surface to volume ratio, GO can adsorb persistent organic pollutants (POPs), such as benzo(a)pyrene (BaP), and act as carrier of POPs, increasing their bioavailability to marine organisms. Thus, uptake and effects of GO in marine biota represent a major concern. This work aimed to assess the potential hazards of GO, alone or with sorbed BaP (GO+BaP), and BaP alone in marine mussels after 7 days of exposure. GO was detected through Raman spectroscopy in the lumen of the digestive tract and in feces of mussels exposed to GO and GO+BaP while BaP was bioaccumulated in mussels exposed to GO+BaP, but especially in those exposed to BaP. Overall, GO acted as a carrier of BaP to mussels but GO appeared to protect mussels towards BaP accumulation. Some effects observed in mussels exposed to GO+BaP were due to BaP carried onto GO nanoplatelets. Enhanced toxicity of GO+BaP with respect to GO and/or BaP or to controls were identified for other biological responses, demonstrating the complexity of interactions between GO and BaP., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

The presence of microplastics in the food chain is a public concern worldwide, and its analysis is an analytical challenge. In our research, we apply Raman imaging to study the presence of 1 μm polystyrene microplastics in cryosections of Mytilus galloprovincialis due to its wide geographic distribution, widespread occurrence in the food web, and general high presence in the environment. Ingested microplastics are accumulated in the digestive tract, but a large number can also be rapidly eliminated. Some authors state that the translocation of microplastics to the epithelial cells is possible, increasing the risk of microplastics transmission along the food chain. However, as seen in our study, a surface imaging approach (2D) is probably not enough to confirm the internalization of particles and avoid misinterpretation. In fact, while some microplastic particles were detected in the epithelium by 2D Raman imaging, further 3D Raman imaging analysis demonstrated that those particles were dragged from the lumens to the epithelium during sample preparation due to the blade drag effect of the cryotome, and subsequently located on the surface of the analyzed cryosection, discarding the translocation to the epithelial cells. This effect can also happen when the samples are fortuitously contaminated during sample preparation. Several research articles that use similar analytical techniques have shown the presence of microplastics in different types of tissue. It is not our intention to put such results in doubt, but the present work points out the necessity of appropriate three-dimensional analytical methods including data interpretation and the need to go a step further than just surface imaging analysis., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Kepa Castro reports financial support was provided by Basque Business Development Agency. Andreas Seifert reports financial support was provided by Ministry of Science Technology and Innovations. Andreas Seifert reports financial support was provided by Basque Business Development Agency. Urtzi Izagirre reports financial support was provided by Ministry of Economic Affairs and Digital Transformation. Urtzi Izagirre reports financial support was provided by Basque Business Development Agency., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Agricultural expansion and intensification are having a huge impact on plant and arthropod diversity and abundance, affecting food availability for farmland birds. Difficult food access, in turn, can lead to immunosuppression and a higher incidence of parasites. In the studies designed to examine changes in the diet of birds and their parasites, metabarcoding is proving particularly useful. This technique requires mini-barcodes capable of amplifying the DNA of target organisms from fecal environmental DNA. To help to understand the impact of agricultural expansion on biodiversity, this study sought to design and identify mini-barcodes that might simultaneously assess diet and intestinal parasites from the feces of farmland birds. The capacity to identify diet and parasites of 2 existing and 3 newly developed mini-barcodes was tested "in silico" in relation to the behavior of a reference eukaryotic barcode. Among the newly designed mini-barcodes, MiniB18S&#95;81 showed the higher taxonomic coverage of eukaryotic taxa and a greater amplification and identification capacity for diet and parasite taxa. Moreover, when it was tested on fecal samples from 5 different steppe bird species, MiniB18S&#95;81 showed high taxonomic resolution of the most relevant diet and parasite phyla, Arthropoda, Nematoda, Platyhelminthes, and Apicomplexa at the order level. Thus, the mini-barcode developed emerges as an excellent tool to simultaneously provide detailed information regarding the diet and parasites of birds, essential for conservation and management., (© 2022 The Authors. Integrative Zoology published by International Society of Zoological Sciences, Institute of Zoology/Chinese Academy of Sciences and John Wiley & Sons Australia, Ltd.)

Chemical signals deposited in feces play an important role in intraspecific and interspecific communication of many mammals. We collected fresh feces of adult wolves from wild breeding groups. All samples visually identified as belonging to wolves were subsequently identified to species level by sequencing a small fragment of mtDNA and sexed typing DBX6 and DBY7 sex markers. Using gas chromatography-mass spectrometry (GC-MS), we identified 56 lipophilic compounds in the feces, mainly heterocyclic aromatic organic compounds, such as indole or phenol, but also steroids, such as cholesterol, carboxylic acids and their esters between n-C 4 and n-C 18 , aldehydes, alcohols and significant quantities of squalene and α-tocopherol, which would increase the chemical stability of feces on humid substrates. There was variability in the number and proportions of compounds between sexes, which could be indicative of their function as chemical signals. We also found variability in different reproductive states, especially in odorous compounds, steroids and α-tocopherol. Feces with a presumed marking function had higher proportions of α-tocopherol and steroids than feces with non-marking function. These compounds could be involved in intragroup and intergroup communication of wolves and their levels in feces could be directly related with the wolf's sex and physiological and reproductive status., (© 2023. The Author(s).)

Global warming and acidification of the global ocean are two important manifestations of the ongoing climate change. To characterize their joint impact on Vibrio adaptation and fitness, we analyzed the temperature-dependent adaptation of Vibrio harveyi at different pHs (7.0, 7.5, 8.0, 8.3 and 8.5) that mimic the pH of the world ocean in the past, present and future. Comparison of V. harveyi growth at 20, 25 and 30 °C show that higher temperature per se facilitates the logarithmic growth of V. harveyi in nutrient-rich environments in a pH-dependent manner. Further survival tests carried out in artificial seawater for 35 days revealed that cell culturability declined significantly upon incubation at 25 °C and 30 °C but not at 20 °C. Moreover, although acidification displayed a negative impact on cell culturability at 25 °C, it appeared to play a minor role at 30 °C, suggesting that elevated temperature, rather than pH, was the key player in the observed reduction of cell culturability. In addition, analyses of the stressed cell morphology and size distribution by epifluorescent microscopy indicates that V. harveyi likely exploits different adaptation strategies (e.g., acquisition of coccoid-like morphology) whose roles might differ depending on the temperature-pH combination.

This investigation deals with how temperature influences oil toxicity, alone or combined with dispersant (D). Larval lengthening, abnormalities, developmental disruption, and genotoxicity were determined in sea urchin embryos for assessing toxicity of low-energy water accommodated fractions (LEWAF) of three oils (NNA crude oil, marine gas oil -MGO-, and IFO 180 fuel oil) produced at 5-25 °C. PAH levels were similar amongst LEWAFs but PAH profiles varied with oil and production temperature. The sum of PAHs was higher in oil-dispersant LEWAFs than in oil LEWAFs, most remarkably at low production temperatures in the cases of NNA and MGO. Genotoxicity, enhanced after dispersant application, varied depending on the LEWAF production temperature in a different way for each oil. Impaired lengthening, abnormalities and developmental disruption were recorded, the severity of the effects varying with oil, dispersant application and LEWAF production temperature. Toxicity, only partially attributed to individual PAHs, was higher at lower LEWAF production temperatures., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Ionan MARIGOMEZ reports financial support was provided by EU HORIZON 2020. Ionan MARIGOMEZ reports financial support was provided by Basque Government. Laure DE MIGUEL-JIMENEZ reports financial support was provided by Ministry of Education and Science., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

5S rRNA is highly transcribed in fish oocytes and this transcription levels can be used to identify the presence of oocytes in the intersex testes of fish exposed to xenoestrogens. Similar to 5S rRNA, tRNAs are transcribed by RNA polymerase III (Pol-III) in eukaryotes, so this study focuses in the analysis of the levels of expression of tRNAs in the gonads (ovaries and testes) of eight teleost species as a possible new oocyte molecular marker. Total RNA extracted from gonads of six commercial teleost species in the Biscay Bay, from the pollution sentinel species thicklip grey mullet (Chelon labrosus) known present intersex testes in response to xenoestrogens in Gernika estuary and from the laboratory model species Danio rerio were analysed through capillary electrophoresis. Bioanalyzer electropherograms were used to quantify the concentrations of tRNAs, 5S and 5.8S rRNA. All studied ovaries expressed significantly higher levels of tRNAs and 5S rRNA than testes. A tRNA to 5.8S rRNA index was calculated which differentiates ovaries from testes, and identifies some intersex testes in between testes and ovaries in mullets. The tRNA/5.8S ratio was highest in ovaries in previtellogenic stage, decreasing towards maturity. Thus, strong oocyte expression of tRNAs is an additional proof of high activity levels of Pol-III during early stages of oocyte development in teleost ovaries. Incidentally, we observed that miRNA concentrations were always higher in testes than ovaries. The indexing approach developed in the present study could have multiple applications in teleost reproduction research and in the development of early molecular markers of intersex condition., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:Ibon Cancio reports financial support was provided by Spain Ministry of Science and Innovation. Ibon Cancio reports financial support was provided by Basque Government., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

In a non-linear system, such as a biological system, the change of the output (e.g., behaviour) is not proportional to the change of the input (e.g., exposure to stressors). In addition, biological systems also change over time, i.e., they are dynamic. Non-linear dynamical analyses of biological systems have revealed hidden structures and patterns of behaviour that are not discernible by classical methods. Entropy analyses can quantify their degree of predictability and the directionality of individual interactions, while fractal dimension (FD) analyses can expose patterns of behaviour within apparently random ones. The incorporation of these techniques into the architecture of precision fish farming (PFF) and intelligent aquaculture (IA) is becoming increasingly necessary to understand and predict the evolution of the status of farmed fish. This review summarizes recent works on the application of entropy and FD techniques to selected individual and collective fish behaviours influenced by the number of fish, tagging, pain, preying/feed search, fear/anxiety (and its modulation) and positive emotional contagion (the social contagion of positive emotions). Furthermore, it presents an investigation of collective and individual interactions in shoals, an exposure of the dynamics of inter-individual relationships and hierarchies, and the identification of individuals in groups. While most of the works have been carried out using model species, we believe that they have clear applications in PFF. The review ends by describing some of the major challenges in the field, two of which are, unsurprisingly, the acquisition of high-quality, reliable raw data and the construction of large, reliable databases of non-linear behavioural data for different species and farming conditions.