Limited Parallelism in Genetic Adaptation to Brackish Water Bodies in European Sprat and Atlantic Herring.

The European sprat is a small plankton-feeding clupeid present in the northeastern Atlantic Ocean, in the Mediterranean Sea, and in the brackish Baltic Sea and Black Sea. This species is the target of a major fishery and, therefore, an accurate characterization of its genetic population structure is crucial to delineate proper stock assessments that aid ensuring the fishery's sustainability. Here, we present (i) a draft genome assembly, (ii) pooled whole genome sequencing of 19 population samples covering most of the species' distribution range, and (iii) the design and test of a single nucleotide polymorphism (SNP)-chip resource and use this to validate the population structure inferred from pooled sequencing. These approaches revealed, using the populations sampled here, three major groups of European sprat: Oceanic, Coastal, and Brackish with limited differentiation within groups even over wide geographical stretches. Genetic structure is largely driven by six large putative inversions that differentiate Oceanic and Brackish sprats, while Coastal populations display intermediate frequencies of haplotypes at each locus. Interestingly, populations from the Baltic and the Black Seas share similar frequencies of haplotypes at these putative inversions despite their distant geographic location. The closely related clupeids European sprat and Atlantic herring both show genetic adaptation to the brackish Baltic Sea, providing an opportunity to explore the extent of genetic parallelism. This analysis revealed limited parallelism because out of 125 independent loci detected in the Atlantic herring, three showed sharp signals of selection that overlapped between the two species and contained single genes such as PRLRA, which encodes the receptor for prolactin, a freshwater-adapting hormone in euryhaline species, and THRB, a receptor for thyroid hormones, important both for metabolic regulation and the development of red cone photoreceptors.

First basin scale spatial-temporal characterization of underwater sound in the Mediterranean Sea.

Anthropogenic underwater noise is an emergent pollutant. Despite several worldwide monitoring programs, only few data are available for the Mediterranean Sea, one of the global biodiversity hotspots. The results of the first continuous acoustic programme run at a transnational basin scale in the Mediterranean Sea are here presented. Recordings were done from March 2020 to June 2021, including the COVID-19 lockdown, at nine stations in the Northern Adriatic Sea. Spatial-temporal variations of the underwater sound are described, having one third octave band sound pressure levels (SPLs) from 10 Hz to 20 kHz as metrics. Higher and more variable SPLs, mainly related to vessel traffic, were found close to harbours, whereas Natura 2000 stations experienced lower SPLs. Lower values were recorded during the lockdown in five stations. Median yearly SPLs ranged between 64 and 95 as well as 70 and 100 dB re 1 µPa for 63 and 125 Hz bands, respectively. These values are comparable with those previously found in busy shallow EU basins but higher levels are expected during a business-as-usual period. This is a baseline assessment for a highly impacted and environmental valuable area, that needs to be managed in a new sustainable blue growth strategy.

Application of an analytical approach to characterize the target strength of ancillary pelagic fish species.

The lack of data on the species-specific Target Strength (TS) on ancillary species limits the application of acoustic surveys in assessing their abundance and distribution worldwide. The TS values of Scomber colias and Trachurus mediterraneus in use in the Mediterranean Sea rely on studies conducted on other species in the Atlantic and Pacific oceans. Nevertheless, the application of backscattering models offers the possibility to overcome the absence of empirical data handling the parameters that most affect the TS. X-ray scans were performed on 82 specimens to get digital representations of the swimbladder and the fish body which were used as input for the application of the Kirchhoff Ray Mode model to measure the TS as a function of frequency and tilt angle. The morphometric differences between the two species produced divergent relative frequency responses and broadband TS patterns. Moreover, comparing the results with one ex-situ experiment, we found a good agreement considering a mean tilt angle of - 10°, standard deviation = 12°. Our results provide the first theoretical insights into the use of backscattering models as a tool to distinguish between species in the Mediterranean Sea by acoustic method, increasing the knowledge of the acoustic reflectivity of ancillary species.

First assessment of underwater sound levels in the Northern Adriatic Sea at the basin scale.

The protection of marine habitats from human-generated underwater noise is an emerging challenge. Baseline information on sound levels, however, is poorly available, especially in the Mediterranean Sea. To bridge this knowledge gap, the SOUNDSCAPE project ran a basin-scale, cross-national, long-term underwater monitoring in the Northern Adriatic Sea. A network of nine monitoring stations, characterized by different natural conditions and anthropogenic pressures, ensured acoustic data collection from March 2020 to June 2021, including the full lockdown period related to the COVID-19 pandemic. Calibrated stationary recorders featured with an omnidirectional Neptune Sonar D60 Hydrophone recorded continuously 24 h a day (48 kHz sampling rate, 16 bit resolution). Data were analysed to Sound Pressure Levels (SPLs) with a specially developed and validated processing app. Here, we release the dataset composed of 20 and 60 seconds averaged SPLs (one-third octave, base 10) output files and a Python script to postprocess them. This dataset represents a benchmark for scientists and policymakers addressing the risk of noise impacts on marine fauna in the Mediterranean Sea and worldwide.

Resource Partitioning among "Ancillary" Pelagic Fishes (Scomber spp., Trachurus spp.) in the Adriatic Sea.

The Mediterranean is one of the most overfished seas of the world where mesopredators are severely threatened. The trophic strategies of four pelagic species that inhabit the Adriatic Sea (Scomber spp. and Trachurus spp.) were investigated through an integrated approach of stomach contents and stable isotopes analyses. Our study demonstrated that Scomber colias feeds mainly on strictly pelagic prey, with fish larvae as a secondary prey in the Southern Adriatic Sea, while S. scombrus feeds on prey belonging to higher trophic levels. Smaller specimens of Trachurus mediterraneus have a diet mainly based on pelagic prey, while larger fishes rely on prey such as benthic decapods, showing an ontogenetic shift in the diet of the species. Trachurus trachurus shows a preference for offshore and deeper areas and a diet such as that of its congeneric, but no clear ontogenetic shift was observed. This spatial segregation allows the co-existence of these two species of Trachurus. Scomber colias mainly inhabits southern areas and S. scombrus shows a preference for the northern sectors. This latitudinal gradient avoids the overlap of their trophic niches. Bayesian mixing models confirmed that the trophic niches of these species only partially overlap in the middle of the trophic web.

Detection of endocrine disrupting chemicals and evidence of their effects on the HPG axis of the European anchovy Engraulis encrasicolus.

Natural/synthetic Endocrine Disrupting Chemicals (EDCs) may display estrogenic activity and a lower potency than 17ß-estradiol. Nonetheless, their concentrations and additive effects can affect the endocrine system and reproductive processes related to the Hypothalamic-Pituitary-Gonadal (HPG) axis. Because of their persistence in both the environment and biological systems, they ultimately target multi-level predators, including humans. We detected presence and effects of xenobiotics on wild anchovy Engraulis encrasicolus in the Western Adriatic Sea. Twenty-one PCBs and five organochlorines were detected on the order of ng g-1; vitellogenin, vitellogenin receptor and genes encoding for the zona radiata proteins were evaluated in gonad and/or liver and found transcribed in male specimens; in addition, intersex was histologically identified in the 13% of testis. Our results have developed the understanding of the European anchovy's reproductive toxicological risk and our approach could assist the comprehension of the complex dynamics of commercially relevant Teleost species.

Characterization and transcriptional profiles of Engraulis encrasicolus' GnRH forms.

The European anchovy Engraulis encrasicolus, a member of the Clupeiformes order, holds a great biological and economical importance. In the past, this species was mostly investigated with the aim of assessing its reproductive biology, trophic ecology, population dynamics and the relations existing with the physical environment. At present days, though, an almost complete lack of information afflicts its neuroendocrinology and reproductive physiology. The hypothalamic-pituitary-gonadal (HPG) axis at its highest levels was herein investigated. In this study, the gonadotropin-releasing hormone (GnRH), a neuropeptide underlying many reproduction-related processes, the most critical of which is the stimulation of gonadotropin synthesis and secretion from the pituitary gland, was cloned. Three forms (salmon GnRH, chicken-II GnRH and the species-specific type) were characterized in their full-length open-reading frames and, in accordance with other Clupeiformes species, the distinctive one was found to be the herring-type GnRH. We qualitatively and semiquantitatively evaluated the localizations of expressions and the temporal transcription patterns of the three GnRH forms in male and female specimens throughout their reproductive cycle as well as described their phylogeny with regard to teleost GnRH lineages, and, specifically, to other Clupeiformes species.

Clupeiformes' Egg Envelope Proteins characterization: The case of Engraulis encrasicolus as a proxy for stock assessment through a novel molecular tool.

Zona radiata proteins are essential for ensuring bactericidal resistance, oocyte nutrients uptake and functional buoyancy, sperm binding and guidance to the micropyle, and protection to the growing oocyte or embryo from the physical environment. Such glycoproteins have been characterized in terms of molecular structure, protein composition and phylogenetics in several chordate models. Nevertheless, research on teleost has not been extensive. In Clupeiformes, one of the most biologically relevant and commercially important order which accounts for over 400 species and totally contributes to more than a quarter of the world fish catch, Egg Envelope Protein (EEP) information exist only for the Clupea pallasii and Engraulis japonicus species. The European anchovy, Engraulis encrasicolus, the target of a well-consolidated fishery in the Mediterranean Sea, has been ignored until now and the interest on the Otocephala superorder has been fragmentally limited to some Cypriniformes and Gonorynchiformes, as well. The aim of the present study was to fill the ZP protein-wise gap of knowledge afflicting the understanding of the European anchovy's reproductive process and to expand the background on Clupeiformes. We cloned the five Engraulis encrasicolus' zp genes and deduced their products, determined their tissue distribution, quantified their mRNA expression throughout the reproductive cycle and provided an insight into their evolution through phylogenetic tools. Furthermore, we proposed a multivariate statistics-based method to objectively infer and/or confirm the classification of Engraulis encrasicolus' sexual maturity stages by analyzing data of zp mRNAs' relative abundance.

Biocomplexity in Populations of European Anchovy in the Adriatic Sea.

The sustained exploitation of marine populations requires an understanding of a species' adaptive seascape so that populations can track environmental changes from short- and long-term climate cycles and from human development. The analysis of the distributions of genetic markers among populations, together with correlates of life-history and environmental variability, can provide insights into the extent of adaptive variation. Here, we examined genetic variability among populations of mature European anchovies (n = 531) in the Adriatic (13 samples) and Tyrrhenian seas (2 samples) with neutral and putative non-neutral microsatellite loci. These genetic markers failed to confirm the occurrence of two anchovy species in the Adriatic Sea, as previously postulated. However, we found fine-scale population structure in the Adriatic, especially in northern areas, that was associated with four of the 13 environmental variables tested. Geographic gradients in sea temperature, salinity and dissolved oxygen appear to drive adaptive differences in spawning time and early larval development among populations. Resolving adaptive seascapes in Adriatic anchovies provides a means to understand mechanisms underpinning local adaptation and a basis for optimizing exploitation strategies for sustainable harvests.

Coupling Demographic and Genetic Variability from Archived Collections of European Anchovy (Engraulis encrasicolus).

It is well known that temporal fluctuations in small populations deeply influence evolutionary potential. Less well known is whether fluctuations can influence the evolutionary potentials of species with large census sizes. Here, we estimated genetic population parameters from as survey of polymorphic microsatellite DNA loci in archived otoliths from Adriatic European anchovy (Engraulis encrasicolus), a fish with large census sizes that supports numerous local fisheries. Stocks have fluctuated greatly over the past few decades, and the Adriatic fishery collapsed in 1987. Our results show a significant reduction of mean genetic parameters as a consequence of the population collapse. In addition, estimates of effective population size (Ne) are much smaller than those expected in a fishes with large population census sizes (Nc). Estimates of Ne indicate low effective population sizes, even before the population collapse. The ratio Ne/Ne ranged between 10-6 and 10-8, indicating a large discrepancy between the anchovy gene pool and population census size. Therefore, anchovy populations may be more vulnerable to fishery effort and environmental change than previously thought.