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Marine diatoms of the genus Pseudo-nitzschia are widespread and occur in various environments. About half of the species described today have been shown to be toxic, producing the potent neurotoxin, domoic acid. The Pseudo-nitzschia species are a common component of the Adriatic phytoplankton community and are occasionally predominant. In the present study, the composition of the Pseudo-nitzschia species was studied in four distinct aquaculture areas from February 2022 to February 2023. These are the first results of a comprehensive study of Pseudo-nitzschia diversity combining morphological and molecular analyses. The integrated approach resulted in the confirmation of Pseudo-nitzschia brasiliana, Pseudo-nitzschia hasleana, Pseudo-nitzschia galaxiae, and Pseudo-nitzschia linea. The species, P. galaxiae, was the most frequently occurring, P. linea was found in all investigated areas, while P. hasleana and P. brasiliana were rare, and their distribution was limited mainly to Kaštela Bay. The findings of P. brasiliana and P. hasleana represent the first confirmation for the Adriatic Sea, while P. galaxiae and P. linea have not been previously reported along the eastern Adriatic coast.

By introduction of DNA barcodes in Adriatic larval fish identification possible presence of new Gobiidae - Buenia massutii Kovačić, Ordines, and Schliewen, 2017, was noticed. Till now, occurrence of this species was restricted only to the Western Mediterranean and to the neighbouring part of the Atlantic Ocean., Larva nedavno opisane vrste Buenia massutii Kovačić, Ordines i Schliewen, 2017, s poznatom geografskom distribucijom ograničenom na zapadno Sredozemlje i susjedni dio Atlanskog oceana, je pronađena u Jadranskom moru. Naime, tijekom ihtioplanktonskog uzorkovanja je izolirana larva navedene vrste, čija je taksonomija utvrđena molekularnom analizom (DNA barcoding).

Within this paper, reproduction traits of the Adriatic population European hake, Merluccius merluccius (Linnaeus, 1758), were investigated. Specimens of the target species were monthly collected (N=1173) during 2019 along the eastern Adriatic Sea with commercial bottom trawlers. Overall, European hake total body length varied between 15.5 and 49.5 cm (mean±SD=27.5±5.0 cm) and domination of females was noted in larger length classes (TL>31.0 cm). The sexual ratio of all specimens was m/f=0.64; male prevalence was established over the year, except during the spawning peaks (December, June) when the sex ratio was in favour of the females. According to European hake’s maturity stages, gonad weights, gonadosomatic indexes and histological analysis of the gonad tissues, this species in Adriatic spawn twice in one year; the spawning period was from December to February, while second smaller signs of spawning were observed at the beginning of the summer, in June. 50% of the European hake male and female specimens sexually matured at 20.7 cm and 22.4 cm of total length, respectively., U okviru ovog rada je istražena reproduktivna biologija jadranske populacije oslića, Merluccius merluccius (Linnaeus, 1758). Uzorci istraživane vrste su prikupljeni iz uzoraka lovina ostvarenih komercijalnom pridnenom mrežom koćom s područja istočnog Jadrana (Hrvatsko ribolovno more) jednom mjesečno (N = 1173) tijekom 2019. godine. Raspon totalnih dužina tijela oslića se kretao između u rasponu od 15,5 cm do 49,5 cm (srednja vrijednost ± SD = 27,5 ± 5,0 cm) te je pri većim dužinskim razredima zabilježena dominacija ženki (TL> 31,0 cm). Omjer spolova (m/ž) je iznosio 0,64; prevalencija mužjaka je utvrđena tijekom čitave godine izuzev razdoblja mrijesta (prosinac, lipanj) kada je omjer spolova bio u korist ženki. Analizom stadija zrelosti, mase gonada, gonadosomatskog indeksa te histoloških preparata tkiva spolnih žlijezda utvrđeno je da se ova vrsta u Jadranu mrijesti dva puta u jednoj godini; utvrđeno je razdoblje mrijesta od prosinca do veljače, dok je drugo razdoblje mrijesta nešto slabijeg intenziteta utvrđeno početkom ljeta, točnije u lipnju. Prva spolna zrelost svih analiziranih jedinki oslića je nastupila pri totalnoj dužini tijela od 20,7 cm za mužjake odnosno 22,4 cm za ženke.

The blue jack mackerel Trachurus picturatus (Bowdich, 1825) specimens (N = 155) were collected during the MEDITS survey, done along the eastern side, precisely, of the Croatian fishing ground in July 2018. Biometrical analysis of ten morphometric and five meristic characters, as well as genetic analysis proved that the collected specimens were blue jack mackerel. The total length (TL) and weight (W) of all observed specimens ranged from 9.2 to 33.7 cm (12.15 &plusmn, 2.95 cm) and from 5.79 to 384.94 g (17.64 &plusmn, 39.42 g), respectively. All calculated length&ndash, length relationships were linear (r &gt, 0.923). Sex was determined only on two larger specimens (28 cm &lt, TL &lt, 32.8 cm), which were females. In the length&ndash, weight relationship, positive allometry was established (b = 3.1789). Based on 37 partial cytochrome b sequences, the overall haplotype diversity (h) of 0.812 &plusmn, 0.048 and nucleotide diversity (&pi, ) of 0.0064 &plusmn, 0.0007 indicated high levels of haplotype and low nucleotide diversity. The obtained sequences were compared to previously published research within the Northeast Atlantic Ocean and the Mediterranean Sea, confirming the absence of genetic structure among these populations.

The Atlantic bluefin tuna (ABFT; Thunnus thynnus) today represents one of the economically most important species for Croatian fisheries industry. Although the most diverse and abundant parasitofauna is usually found in the largest specimens of wild ABFT, the opposite was observed in captivity where parasite populations significantly decline by the end of the farming cycle. Copepod Brachiella thynni, is a skin parasite frequently parasitizing tuna, whose population also decreases in number throughout the rearing process. In order to better understand the immunity mechanisms underlying ABFT reaction to B. thynni infection, we studied expression profiles of immunity related genes; interleukin 1β (il1β), tumour necrosis factors (tnfα1, tnfα2), complement component 4 (c4) and caspase 3 (casp3), in peripheral blood leukocytes (PBLs) during in vitro stimulation by B. thynni protein extracts (i.e. antigens) and in infected tissues at B. thynni parasitation site. Finally, a histopathological analysis of semi-thin and ultra-thin sections of tissues surrounding B. thynii attachment site was performed to evaluate the severity of parasite-induced lesions and identify involved cell lineages. In vitro stimulation of ABFT PBLs with B. thynii antigens caused a dose-depended upregulation of selected genes, among which tnfα1 showed the highest induction by both concentrations of B. thynni protein extract. However, targeted genes were not significantly upregulated in the infected tissue. Also, no significant alterations in ultrastructure of epithelial layers surrounding B. thynii attachment site were noticed, except local tissue erosion, necrosis of squamous epithelium and proliferation of rodlet and goblet cells. Our results suggest that B. thynii has evolved strategies to successfully bypass both innate immune response and the connective-tissue proliferation processes. Therefore, the observed disappearance of this copepod by the end of the rearing process is more likely related to its limited lifespan on the host and its inability to complete the life cycle in the rearing cages, rather than host's reaction.

In gilthead seabream the number of domesticated individuals increased annually, and escape events occur regularly in the Adriatic Sea. Still there is a lack of population genetic characteristics and evidence of the extent and geographic scale of interbreeding resulting from fish-farm escapees. We screened 1586 individuals using a panel of 21 neutral microsatellite loci in several consecutive years and here report on the medium-scale detection of hybrid and farmed seabream in the natural environment. Wild adults showed a lack of genetic structure within basin and sampling years and reduced connectivity with wild offspring collection, suggesting their temporal residency within the Adriatic. On the contrary, by linking the results of multiannual genetic analyses with the results of coupled hydrodynamic and individual based models (IBM-Ichthyop), we observed a strong connection of wild seabream associated with tuna-aquaculture sites and offspring from the nursery grounds, indicating that the surroundings of tuna sea-cage farms can function as a spawning grounds. The study results present the genetic baseline of wild and farmed strains from the eastern Adriatic Sea, as a first step toward development of a mitigation strategy for fish escapees aimed at controlling further erosion of genetic integrity.

The spawning behavior of captive Bluefin Tuna (Thunnus thynnus) was observed in cages at an Adriatic facility in summer 2011. Approximately 20,000 eggs were collected from cages and transferred to a land-based nursery, with an estimated fertilization success rate of 80%. Eggs were spherical with a diameter of 1,035.06 ± 20.16 μm and were pelagic with a single oil globule (227.63 ± 8.07 μm). Newly hatched larvae (3.08 ± 0.14 mm total length) followed scombrid larval development: melanophores were scattered over the body, head, notochord, and yolk, except finfold; eye pigmentation was observed 1.5 days posthatch, pectoral fins started to appear, and two-thirds of the yolk sac were absorbed; the mouth developed at 2.5 days posthatch, and larvae began feeding upon enriched rotifers. After mortalities occurred in the nursery (5 days posthatch), DNA was extracted from a sample of larvae. An 890 base pair fragment of the mitochondrial partial control region was amplified and sequenced to genetically con...

A multixenobiotic resistance (MXR) mechanism mediated by ABC binding cassette (ABC) transport proteins is an efficient chemical defence mechanism in sea urchin embryos. The aim of our work was to evidence whether exposure to sub- lethal doses of specific contaminants (oxybenzone (OXI), mercuric chloride (HgCl2), trybutiltin (TBT)) would induce MXR transporter activity during embryonic development (from zygote to blastula stage) in purple sea urchin (Paracentrotus lividus) embryos. Further, we present data on molecular identification, transport function, expression levels and gene localisation of two ABC efflux transporters – P- glycoprotein (ABCB1/P-gp) and sulfonylurea receptor like protein (ABCC9/SUR-like). Partial cDNA sequences of abcb1 and abcc9 were identified and quantitative PCR (qPCR) evidenced an increase in mRNA transcript levels of both ABC transporters during the 2-cell, as well as an overall decrease during the blastulae stage. Calcein- AM efflux activity assay indicated the activation of multidrug resistance-associated protein (MRP)/ABCC-like transport in the presence of HgCl2 and TBT in exposed blastulae. The in situ hybridisation of the 2-cell and blastula stages showed ubiquitous localisation of both transcripts within cells, supporting qPCR data. In conclusion, ABCB1 and ABCC9 are constitutive, as are HgCl2, TBT and OXI- inducible ABC membrane transporters, coexpressed in the zygote, 2-cell and blastula stages of the P. lividus. Their ubiquitous cell localisation further fortifies their protective role in early embryonic development.

Knowledge concerning cetacean ecology in the Mediterranean is limited but important for sustainable planning and enforcement of appropriate conservation measures. Any information that might help to elucidate their ecology is essential. We explored the population and genetic structures of Anisakis spp. nematodes isolated from four toothed whale species – bottlenose dolphins (Tursiops truncatus), striped dolphins (Stenella coeruleoalba), Risso’s dolphins (Grampus griseus) and Cuvier’s beaked whales (Ziphius cavirostris) – stranded along the eastern Adriatic Sea coast (1990–2012) to reveal more information on host ecological patterns. Lower parasite prevalence was observed in resident dolphin species compared with occasionally occurring species, as well as in young compared with adult dolphins, indicating different feeding habits related to age. No unequivocal relationship between the biological traits of a host (age, body length, body mass and blubber depth) and Anisakis population parameters was observed. Phylogenetic analysis revealed a new geographical record of Anisakis simplex sensu stricto (1.96%) and Anisakis physeteris (1.31%) in the Adriatic Sea in addition to resident Anisakis pegreffii (96.73%). In an assessment of the Adriatic Sea and oceans worldwide, the genetic structure of Anisakis revealed that A. pegreffii populations do not differ among various final host species but do differ with respect to geographical location in contrast to previously accepted Anisakis panmixia.

Pro-inflammatory cytokines play an important role in teleost defence against numerous types of pathogens, therefore are often used as biomarkers during various infections. In order to evaluate Atlantic bluefin tuna IL-1β, TNFα1 and TNFα2 induction by PAMPs, we quantified their expression during in vitro stimulation of peripheral blood leukocytes with LPS, Poly I:C. Furthermore, their role in acute and chronic parasitic infection was examined during natural infection of Pseudocycnus appendiculatus (Copepoda) and Didymosulcus katsuwonicola (Digenea), as well as during leukocyte exposure to total protein extracts isolated from two parasite species. Induction of ABT IL- 1β and TNFα2 by PAMPs and protein extracts from D. katsuwonicola and P. appendiculatus, as well as during natural infection with two parasites, suggests these cytokines play an important role in inflammation, being engaged in controlling parasite infections, in contrast to ABT TNFα1. Cellular innate response to the digenean D. katsuwonicola showed rather chronic character, resulting with parasite encapsulation in connective tissue. Mast cells, eosinophils, goblet cells, and occasional rodlet cells found at the site of infection, along with the induction of TNFα2, suggest the presence of a moderate inflammatory reaction that fails to seriously endanger digenean existence. In contrast, copepod P. appendiculatus, attached to the gill epithelium by clamping, caused direct tissue disruption with undergoing necrotic or apoptotic processes, and extensive proliferation of rodlet and goblet cells. Differential expression patterns of target cytokines in tissue surrounding two parasites and in vitro PBL model suggest that quality and quantity of tuna immune response is conditioned by parasite adaptive mechanisms and pathogenicity.

Atlantic bluefin tuna (BFT) (Thunnus thynnus) is of great economic significance for world aquaculture and therefore it is necessary to ensure optimal and sustainable conditions for the farming of this species. Intensive culture of fish may be limited by infectious diseases that can impact on growth performance and cause heavy losses. However, to date there are no reports of cloning and expression analysis of any major immune genes of Atlantic BFT although some immune genes are known in other BFT species. Therefore the aim of this study was to characterize the first cytokine molecules in Atlantic BFT, through: 1) Isolation of full-length cDNA and gene sequences of TNFα1, TNFα2 and IL-1β, 2) comparison of these molecules to known sequences in other vertebrates, especially teleost fish, by multiple sequence alignment, phylogenetic tree analysis and homology modeling ; 3) Quantification of in vivo expression of these cytokines in selected tissues in reared BFT over the duration of the farming process. The results indicated that these three cytokines could have value for monitoring Atlantic BFT health status. Curiously, the liver seemed to be an important site of cytokine production during poor health conditions in this species, perhaps reflecting its role as an important organ involved in fish defenses.