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Tremoctopods are epipelagic argonautoid octopods characterized by their expanded dorsal webs and strong sexual size dimorphism, with dwarfed males. The scarcity of taxonomic features attributed to this genus presents a challenge, and there is growing evidence of species misidentification in Tremoctopus genus on a molecular level. In this study, we investigated four female specimens of blanket octopus Tremoctopus violaceus caught by purse seine fishing in the Central Eastern Adriatic Sea in 2019. Individuals had smooth, firm and muscular bodies, dark bluish purple on the dorsal and iridescent silvery on the ventral side, with dorsal mantle lengths of 113, 82, 80 and 78 mm. The constructed phylogenetic trees based on the 16S ribosomal RNA and cytochrome c oxidase subunit I sequences of investigated Adriatic specimens and publicly available sequences showed strong support for the T. violaceus clade, consisting of individuals collected from the Adriatic Sea, Mediterranean Sea and Atlantic Ocean, with the exclusion of Indo-Pacific clade most probably corresponding to T. gracilis. To fully understand the life-history traits of Tremoctopus species, future research should focus on DNA-based methods for correct species identification combined with morphological characters, geographic distribution and ecological information.

Taxonomic position of the species Troglaegopis mosorensis (Riedel & Rađa, 1983) (Kuščer, 1933), based on shell morphology and anatomy of reproductive organs place this species in a family Zonitidae. Shell morphology is very similar to representatives of family Zonitidae but “brachium copulatorum” ( very long vaginal appendage) is a characteristic for family Vitrinidae. In our survey we combined morphology-based taxonomy and molecular techniques to define the current taxonomic status of T. mosorensis whose internal morphology (genital organs) bear some characteristics of the family Vitrinidae (Riedel and Radja 1983 ; Hausdorf 2002), but the species is included in the family Zonitidae. In order to determine whether species Troglaegopis mosorensis belongs to the family Zonitidae or Vitrinidae we performed a Maximum Likelihood and Bayesian phylogenetic analysis by combining our data and data on the species of the genus Zonites (Montfort, 1810) and Turcozonites (Riedel, 1987) from GenBank. We have determined the mitochondrial 16S rDNA, COI, H3 and ITS region sequences of 21 Troglaegopis, 4 Meledella, 6 172 Aegopis and 7 Vitrina specimens. Our results indicate the separate position of the genus Troglaegopis and unexpectedly A. verticillus in relation to the families Zonitidae and Vitrinidae that suggest new systematic position on family level for the genera Troglaegopis and Aegopis.

Bacterioplankton represent major constituents of marine food webs and participate in biogeochemical cycles of all major elements essential for life. They are metabolically and taxonomically diverse community strongly shaped by environmental factors (temperature, salinity, nutrients, predation). The biodiversity and seasonality of different bacterial groups was assessed along a trophic gradient in the eastern Adriatic Sea during one year of monthly sampling performed within ADRISAAF project. Sampling included three stations: a near shore coastal location in the Kaštela bay near Split, Croatia ; a semi-open canal and an oligotrophic location near the island of Vis. The structure of bacterial communities was assessed by catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH). Preliminary results suggest a seasonal and spatial trend in bacterial diversity and community structure. One of major community constituents was the clade SAR11 with increased abundance during spring in euphotic zone of eutrophic waters, and deeper layers during winter in oligotrophic waters. Bacterial communities were characterized by Bacteroidetes and Gammaproteobacteria in deeper layers of oligotrophic waters and Alphaproteobacteria in surface layers of eutrophic waters. The Roseobacter group, physiologically and genetically versatile clade within Rhodobacteraceae (Alphaproteobacteria), increased their abundance in spring and autumn in surface to 30 m deep waters of coastal areas. The results are discussed and compared to environmental variables. These data represent the first comprehensive analysis of major bacterial groups in the Eastern Middle Adriatic Sea aiding in the understanding of the food web dynamics in the investigated ecosystem.

Aerobic Anoxygenic Phototrophs (AAPs) are facultative photoheterotrophs that are widespread in various habitats and may account for up to 25% of the total marine bacterial population. This functional group is characterized by a high growth rate and significantly larger cells compared to other heterotrophic bacteria, representing important prey in trophic web and a significant proportion of biomass pumped into biogeochemical cycles. Albeit, they are not fully understood and characterized. Based on pufM gene analysis, a marker for AAP light-harvesting centers, they are divided into 12 different phylogroups. The goal of ADRISAAF project is to determine the temporal and seasonal dynamics of AAPs and their contribution to the total carbon budget in the water column of the Middle Adriatic Sea. Sampling was carried out monthly during one year by research vessel Bios Dva, encompassing stations with diverse physicochemical features and trophic status. Bacterial production was determined and AAP population investigated with Infra-Red Fluorescence In Situ Hybridization (IR-FISH) and metabarcoding of pufM gene. Preliminary results reveal species richness and the taxonomic composition of the total AAP community as well as the abundance of Alphaproteobacteria, Gammaproteobacteria, and Roseobacter phylogroups in the Adriatic Sea and their correlation with important ecological factors shaping the community on the spatial and temporal scale. These data represent the first comprehensive analysis of the AAP bacterial community in the Middle Adriatic aiding in the understanding of the carbon flux toward the higher trophic levels in the investigated ecosystem.

In September of 2020, 23 juveniles of neon flying squids were collected in the central eastern Adriatic Sea (the island of Palagruža (N=14), the island of Jabuka (N=1) and the Korčula Channel (N=8)), presenting rare opportunity to study these cephalopods. The dorsal mantle length of the specimens ranged from 65 to 152 mm and body weight from 7.6 to 89.8 g. The partial sequence of the mitochondrial cytochrome c oxidase subunit I (COI) gene was determined and phylogenetic analyses confirmed that collected squids were Ommastrephes caroli (Furtado, 1887). The genus Ommastrephes d'Orbigny has been for many years considered a monotypic cosmopolitan genus with a discontinuous distribution, however recently four species have been erected within this genus using molecular tools. So far, there were only four reported records of this species in the Adriatic Sea. The analyzed individuals shared similar haplotypes as those from both the Mediterranean Sea and North Atlantic. The analysis of statolith growth increments estimated the age of individuals to range from 36 to 71 days (average of 49.3 days). The results of back-calculation indicate that the spawning of this species occurs during the warmer, spring-summer period. Considering the size of caught individuals, Adriatic Sea could represent a potential feeding ground for this species.

Two Allotheuthis species have historically been recorded in the Mediterranean Sea and Adriatic basin: A. media and A. subulata. Their identification has largely been descriptive in nature and conducted using morphometric parameters that have proven insufficient and unreliable in the advent of genetic analyses. In this study we have collected samples of loliginids during two consecutive MEDITS expeditions in 2014 (n=32) and 2015 (n=21) covering entire eastern Adriatic Sea. Specimens were caught using the bottom trawl net and frozen immediately on board until laboratory inspection when nine morphological traits were measured for each specimen and samples taken for DNA processing. Using partial sequencing of cytochrome oxidase I (COI) mtDNA gene, 30 individuals were identified as A. media and 21 as A. subulata, with A. media distributed throughout the basin and A. subulata only in its central and southern parts. Alloteuthis media showed high genetic diversity (Hd = 0.88) and statistically significant genetic differentiation between Adriatic, Aegean and Ionian populations in respect to Atlantic haploytpes, as was inferred by the use of publicly available sequences. Alloteuthis subulata in the Eastern Adriatic shared a single haplotype. Analyses of morphometric data suggested that there was no single morphometric character with strong enough power to discriminate between the two species, however, when morphological traits were looked as a composite metric rather than in isolation, 87.5% of individuals were correctly classified as A. media males, A. media females or A. subulata.

Knowledge of genetic diversity and population structure is the foundation of responsible and sustainable exploitation of every species, that ensures the sustainability of wildlife populations. In order to gain insight into the recent state of genetic diversity of Loligo vulgaris populations in the north, central and south part of the Eastern Adriatic, a part of the nucleotide sequence of the mitochondrial gene of the cytochrome c oxidase subunit I, known as COI, was analyzed. This genetic marker today represents the basis for the identification of all Metazoa, the study of their phylogenetic relationships as well as population structures. During the autumn- winter period of 2019, a total of 30 squids were collected. Sequencing of mitochondrial DNA fragments produced good quality results. The length of the sequenced region in most samples was about 630 base pairs (bp). A total of 9 polymorphic sites and 10 haplotypes were identified in the total sample of L. vulgaris. Of the 9 polymorphic sites, 5 were singletons with two possible variants. Of the 4 phylogenetically informative sites, all had two variants. Of the 10 haplotypes defined, 5 are represented by a single individual. Haplotype 1 (H1) showed the highest total frequency in the total sample. According to genetic diversity indices, high total haplotype diversity (Hd = 0.8005) and low nucleotide diversity (Pi = 0.00199) were found. The total mean difference between sequence pairs (k) was 1.26108. Obtained results indicate that there is a unique homogeneous stock of L. vulgaris in the Adriatic Sea.

Anisakis pegreffii is a nematode that parasitizes marine cetaceans as definitive hosts, accidentally infecting humans for a limited time span. Although the knowledge of disease mediators during anisakiasis in humans is incomprehensive, we can close the gaps by applying what is known from other helminth infections. Cathepsins are key virulence factors in disease progression and synchronized expression of these proteolytic enzymes is considered a hallmark of parasitosis. Figuring as potential disease biomarkers, they are also important subjects in drug or vaccine targets investigation. The functional insights of these proteases vary during life cycle of parasites in context of infection type, eclosion, feeding and immune evasion. Cathepsins are clusters of proteolytic enzymes of lysosomal origin divided into three classes – cysteine, serine and aspartic proteases. Cysteine cathepsins occur in multiple forms as cathepsin B-like (B, O, C, and X or Z), cathepsin L-like (L, V, K, S, and H), cathepsin F-like (W and F), and placenta-specific cathepsins (cathepsins J/P, M, Q, R, 3, 6, 7 and 8) that are exclusively expressed in rat/mouse placenta. Biochemically, cathepsins A and G have been distinguished as serine proteases, whereas D and E belong to aspartic endopeptidases. Through in silico data-mining of A. pegreffii infective third-stage larvae (L3) transcriptomes, we have identified multiple signatures of cathepsin A carboxypeptidases and cathepsin D. Apart from these, L3 express a vast array of cysteine proteases. In addition to cathepsin B, F and L, the presence of putative cathepsin Z and W has also been validated. A. pegreffii presents at least four distinct clades of cathepsin B and L proteolytic enzymes, and a unique cathepsin F having 51% identity to human orthologue. In general, A. pegreffii cysteine cathepsin orthologues share maximum identity to Toxocara canis rather than free-living model Caenorhabditis elegans. Putative A. pegreffii cathepsin B1 has duplet histidine conserved in occluding loop, thus suggesting both effective exopeptidase and endopeptidase activities on specific substrates. Interestingly, the three other putative cathepsin B clades (B2, B3, B4) show to be non-canonical, expressing shorter occluding loops. Such atypical proteases could open interesting dimensions in understanding nematode biology and scoping for drug discovery.

Human anisakiasis is a disease caused by infective larvae of the genus Anisakis spp. (Anisakidae, Nematoda), eliciting gastric, intestinal, ectopic or gastro-allergic form, or eventually an asymptomatic form within the Anisakis-seropositive population. Analysing differentially expressed genes (DEGs) after Illumina RNAsequencing of A. pegreffii third-stage infective larvae (L3) experimentally infecting rat (accidental host, model for human infection) and fish (paratenic host), we identified transcripts that expressed the highest upregulation common for L3 migrating through both hosts. From the initial 65 transcripts, deemed virulence factors (VFs), we selected those with at least 2FC expression, discarded constitutive cuticle elements and non-annotated transcripts, obtaining a list of several catalysts and transporters, already recognised as excretory/secretory products. These included five targets: cytosolic non-specific dipeptidase (CNDP2), leukotriene A-4 hydrolase (LKHA4), aspartic protease 6 (ASP6), ATP-binding cassette sub-family B member 9 (ABCB9), and UDPglucuronosyltransferase (UGT3). We searched VFs against the Wormbase Parasite protein BLAST database, obtained predicted proteomes of selected Nematoda (Clades III, V, IV, C, I) and Platyhelminthes (Clades Monogenea, Trematoda, Cestoda, Rhabditophora), determined homologs of A. pegreffii VFs, and identified single-copy orthologous groups (OGs) in selected proteomes (OrthoFinder v 2.5.2). Finally, we determined VFs 3D structures and catalytic sites utilizing online modelling techniques and comparing our models to structures co-crystalized with inhibitors or substrate analogues. Phylogenetic analyses inferred the presence of four families (ABCB9s, ASP6s, LKHA4s and CNDP2s) in almost all Nematoda, Platyhelminthes and Metazoa examined, and the lack of UGT3 in Trematoda and Cestoda. All VFs showed high levels of duplication and widespread occurrence in closely related Toxocara canis, Ascaris suum, Parascaris univalens and H. contortus, supporting their vital biological functions in nematodes. VFs tertiary structure predictions and modelling analyses showed to be useful for the search of currently available inhibitor molecules, being also applicable in a screening of broad-spectrum efficacy for all Clade III and V nematodes examined

Anisakiasis is considered one of the most important foodborne parasitic zoonoses, although the nematode cannot complete its life cycle in humans. Generation of -omics data has enabled more versatile approached to study host-parasite interactions. To scrutinise transcriptomic signature of Anisakis pegreffii infective larvae (L3) during migration through a oikilotherm (fish, paratenic host) and homeotherm accidental host(rat, model for human infection), we RNA-sequenced (Illumina) isolated L3, performed a differential analysis of gene expression (DEGs) and calculated the enrichment of GO terms and KEGG pathways within DEGs sets. We also differentiated between transcripts of successfully migrating L3 vs those that were expelled through faeces from the host (non-migrating). The final transcriptome, filtered for transcripts with a detectible coding sequence, resulted in 36, 201 transcripts with predicted ORFs and 4, 016 proteins represented by nearly fulllength transcripts and >80% alignment coverage. Of these, 1, 937 were found ifferentially expressed (FDR < 0.05) in migrating vs. non-migrating L3 in rat, Rattus norvegicus (1, 096 up and 841 down), 484 in sea bass, Dicentrarchus labrax (328 up and 156 down) and 509 were differentially regulated in the two hosts. The largest difference in log2FC between migrating L3 in rat and in sea bass was observed for putative cuticle collagen (CO155) and glucose-6-phosphate exchanger (G6PT1), which were both upregulated in rat and downregulated in sea bass, while the opposite was observed for NADH-dependent flavin oxidoreductase (NADA). In the homeothermic accidental host (rat), Anisakis L3 upregulated ribosome-related genes, cell division, cuticle constituents, oxidative phosphorylation, probably in an unsuccessful effort to moult to the next developmental stage. Contrary, in the paratenic poikilotherm host (fish), L3 moderately upregulated or silenced the main metabolic pathways, preparing for dormancy by triggering autophagy and longevity pathways. We hypothesise that the failure of L3 to survive in the accidental host is a result of metabolic/ energy exhaustion that occurs from undetermined migration towards a suitable niche that is never to be achieved, additionally stimulated by host homeothermy. In the poikilotherm paratenic host, moderately upregulated or silenced metabolism serves to enable larvae to prepare for paratenesis, warranting their survival.

Background: Anisakiasis, a zoonotic disease caused by Anisakis spp. larvae, represents a rising public health problem. Presentation of the disease is variable with unspecific gastrointestinal and/or allergic symptoms, accounting for the high number of misdiagnosed cases. Objectives: To better understand the onset of anisakiasis in Sprague-Dawley rats experimentally infected with Anisakis pegreffii, we aimed at characterising early cellular and molecular (6-72 h p.i.) immune response in affected tissues and general regulatory mechanisms. Materials and Methods: Two experiments were performed on 35 and 10 male rats, respectively, and each animal was intubated by the gastric probe with 10 live A. pegreffii larvae or 1.5 mL of saline (external control). At predefined time points, animals were sacrificed and tissues with visible lesions were processed for i) Illumina NextSeq 500 paired-end sequencing, ii) target genes and microRNA expression analysis, iii) histopathological evaluation (HE, IHC, TEM), and iv) global DNA methylation quantification. Results: In total, there were 1372 differentially expressed (DE) genes in the Anisakis-infected rat stomach tissues and 1633 DE genes in the muscle tissues. Il6, Il1b, and Ccl3 showed particularly strong expression in the stomach and visceral adipose tissues. In total, three microRNAs were differentially expressed. Histopathology revealed severe inflammatory/haemorrhagic lesions in stomach tissues, dominated by neutrophils and macrophages. Different numbers of CD3+, CD4+, CD68+, iNOS+ and S100A8/A9+ cells were found in stomach, intestine, and muscle tissues. TEM revealed the presence of eosinophils in the inflammatory infiltrate with a lack of mast cells and diffuse areas of tissue necrosis, particularly in muscle tissues. No changes in global DNA methylation were observed between infected and non-infected tissues. Conclusions: Anisakis infection induces strong immune responses in infected rats with marked induction of specific proinflammatory cytokines and miRNA expression, which seems to favour the activation of the interleukin 17 signalling pathway and the development of the T helper-17 response.

Ekosustavni pristup prepoznat je kao značajan način ocjene stanja ribolovnih resursa kao i stanja morskog okoliša u cjelini. Jasno je da su svi morski organizmi neodvojivi dio ekosustava i snažno ovisni o stanju populacija u hranidbenom lancu kao i o faktorima koji vladaju u okolišu. Dugoročno održivo upravljanje ribarstvom trebalo bi se bazirati na ekosustavnom pristupu što u praksi nije slučaj te se u većini područja zasniva na procjeni stanja pojedinačnih vrsta što se često pokazalo neučinkovitim. Slična je praksa i u Jadranskom bazenu, ali inicijativa Interreg Italija-Hrvatska u sklopu projekta FAIRSEA (Fisheries in the Adriatic Region - a Shared Ecosystem Approach) mogla bi biti prekretnica u upravljanju ribarstvom na ovom području. Projekt okuplja 12 partnerskih institucija i široku bazu dionika. Glavni cilj projekta je razvoj integrirane platforme za ekosustavni pristup ribarstvu sa svim raspoloživim informacijama koje će se koristiti za testiranje, modeliranje i planiranje održive ribarstvene prakse. Platforma obuhvaća elemente poput cirkulacije vodenih masa, planktonske produkcije, rasprostranjenosti glavnih resursa, ulove i kapacitete flota, prostornu raspodjelu napora, preferentno modeliranje, dinamiku hranidbenih mreža, socio- ekonomske utjecaje te inpute dionika. Nadamo se da će ovaj prvi korak postepeno dovesti do šireg razumijevanja značaja ekosustavnog pristupa kao i promjene perspektive o kvalitetnom upravljanju ribarstvom u Jadranskome moru.

Introduction: Nematodes of the genus Anisakis Dujardin, 1845 have an indirect life cycle and their life-stages are propagated through trophic webs of their marine hosts. About 200 marine fish species and 25 cephalopod species are confirmed as Anisakis spp. paratenic or secondary intermediate hosts. Previous research suggested that excretory/secretory Anisakis simplex products could have immunomodulatory role in fish hosts. The aim of this study was to assess the immunogenic potential of Anisakis pegreffii crude extract (CE) in European sea bass (Dicentrarchus labrax) peripheral blood leukocytes (PBLs). Methodology: The experiment was performed on in vitro stimulated PBLs of sea bass, prepared using 51% iso-osmotic Percoll solution, seeded in 6-well plates with L-15 medium, supplemented with 15% FCS and 1% penicillin/streptomycin. Two hours (h) after settlement of PBLs (final concentration of 107 cells/ml), 5 μg/ml of previously prepared A. pegreffii CE was added to test wells. In total, 18 replicates were collected 1 and 12 h post-stimulation and preserved for RNA isolation, including control wells. Illumina NextSeq 500 was used for paired-end sequencing of total of 7 pooled PBLs samples prepared from test and control treatments. Results: After quality control, filtering and mapping, differential expression (DE) analysis of sea bass PBLs revealed 195 (60 down and 135 up- regulated) DE transcripts 1 h post-stimulation and 968 (206 down and 762 up-regulated) DE transcripts 12 h post-stimulation with A. pegreffii CE. Strong and significant fold changes (LogFC >> 1), with steady up-regulation as the experiment progressed, was especially noted for transcripts of pro-inflammatory cytokines (interleukin-1 beta and tumor necrosis factor alpha), as well as interleukin-27 subunit beta, known for its diverse roles in innate immunity. Conclusion: Overall, the most frequent gene ontology terms associated with DE transcripts were positive regulation of cell proliferation, response to stimulus, immune response, and chemokine activity. These results help explain the nature of interaction between sea bass PBLs and A. pegreffii CE.

Introduction: Ribosome biosynthesis (RB) is orchestrated by nucleolus whose main function is rapid production of small and large ribosome subunits. Nucleolus is also essential in the sensing of stress stimuli that target ribosome biogenesis ; a condition known as nucleolar or ribosomal stress. It is exhibited through expression of several p53-dependent or p53- independent response pathways that maintain cell homeostasis. Our aim was to characterise molecular mechanisms and architectural changes in the nucleolus of the Anisakis-stimulated rat peripheral blood leukocytes (PBLs). Methodology: In vitro A. pegreffii crude extract-stimulated Sprague-Dawley rat PBLs were harvested 1 and 12 h post-stimulation for RNA isolation. Illumina NextSeq 500 was used for paired-end sequencing of 7 pooled PBLs. Markers of ribosomal biogenesis (Tp53, CDKN1, CCND1, mTOR, RPL5, RPL11, RPL23, NFE2L2) and inflammation (Il4, Il6, Il7, TNF-α) were measured by qPCR, while nucleolar architecture was assessed by localisation of nucleophosmin, NOP58 and coilin by confocal microscopy. Results: Differential expression (DE) analysis of rat PBLs revealed 53 (22 down and 31 up-regulated) DE transcripts 1 h post- stimulation, whose strong and significant fold changes (LogFC >> 1) were especially noted for transcripts involved in regulation of protein complex assembly, membrane polarisation, mitotic cell cycle and calcium ion binding, to name only the few. In contrast, 39 (5 down and 34 up- regulated) DE transcripts were observed 12 h post-stimulation, mainly involved in proinflammatory functions. Strong scattering of nucleophosmin from nucleolus into the nucleus was evidenced, while nucleolus granular component and Cajal bodies remained mostly unaffected. Conclusion: Transcriptomics, targeted gene expression and confocal microscopy support the hypothesis that A. pegreffii CE in PBLs trigger the onset of nucleolus functional and morphological cues toward increased ribosomal biogenesis already 1 h post-stimulation, enriched in transcripts of 40S and 60S ribosomal proteins. This suggests that nucleolar rearrangements occur early and are soon balanced in order to provide an efficient proinflammatory response to the nematode.

Global migration and trade of the foodstuff, climate change, novel trends in human eating habits characterised by an elevated demand for raw food, are considered as major reasons why reports of food borne infections, especially zoonotic parasitosis, have increased in the last decade. Consequently, health authorities and fishing industry have increasingly become aware of the significance of nematode genus Anisakis, which affects human health and the commercial value of fish products. Adriatic fish, marketed fresh, frozen, salted or marinated and exported to EC, are frequently rejected on the border because of Anisakis infection. Although considered one of the most significant emerging food-borne zoonosis, anisakiasis in many Mediterranean countries is still misdiagnosed or underestimated. During a four-year research within the project AnGEl (Anisakis spp: Genomic Epidemiology) funded by the Croatian Science Foundation, we have assessed Anisakis using a multidisciplinary approach, studying the fish as paratenic, cetaceans as the final and men as accidental host. The project resulted in new knowledge on: i) molecular geo-distribution of Anisakis spp. and its hybrids in the Adriatic Sea fish inferred by a newly developed genome-derived small sequence repeats (SSR) tool ; ii) the histopathological effect of the nematode in the digestive tract of the paratenic, final and the model accidental host inferred by IHC and TEM ; iii) the nematode transciptomic signature during migration through the model host using RNAseq and microRNA quantification ; iv) traits of the larval multixenobiotic resistance mechanisms that will help to design more effective therapy ; v) anti-Anisakis IgE seroprevalence in human population professionally related to the fish processing industry. The most important results will be presented, aiming to trigger better conscience and take home message about this emerging and highly under- and missdiagnosed zoonosis.

Introduction: Anisakis spp. are marine nematodes which can cause zoonotic infection in humans if accidentally ingested alive in raw/undercooked fish or cephalopod meat. The aim of this study was to explore the transcriptomes of Anisakis pegreffii L3 larvae in two remarkably different infection systems, an evolutionary familiar and unfamiliar host. Methodology: Experimental infection was performed on European sea bass (Dicentrarchus labrax) (N = 24, sampling 4, 6, 10, and 12 h post-infection), representing a typical paratenic host, and Sprague-Dawley rats (N = 35, sampling 6, 10, 18, 24, and 32 h post-infection), representing a mammalian “novel-host” model, simulating accidental human infection. Larvae were collected while penetrating various host tissues in active migration or in passive transport through gastrointestinal tract. Samples of extracted RNA from larvae collected from both hosts were paired-end sequenced using Illumina NextSeq 500. Results: In total, there were 132 (69 up and 63 down) differentially expressed (DE) transcripts in migrating compared to non-migrating A. pegreffii larvae in sea bass, out of which 110 had logFC > |1|. Biologically significant DE transcripts were included in carbohydrate transport and metabolism, translation, ribosomal structure and biogenesis, cell cycle control, cell division energy production and conversion, to name a few. In rats, there were 2799 (1606 up and 1193 down) DE transcripts in migrating compared to non-migrating A. pegreffii larvae, out of which 814 had logFC > |1|. In addition to cell cycle control, cell division, energy production and conversion, which were mentioned in larvae from sea bass, biologically significant DE transcripts in larvae from rats were also included in intracellular trafficking, secretion, and vesicular transport, secondary metabolites biosynthesis, transport and catabolism. Conclusion: Studies of transcriptomes of parasites give insights into aspects of gene expression, regulation and function and they represent a significant step in understanding parasites’ biology and interactions with their hosts and disease. Use of transcriptomics to reveal mechanisms of interactions of Anisakis and its paratenic and accidental host is applied for the first time in this study.

Global migration, changes in dietary habits such as eating raw or undercooked food, higher demand and availability of exotic food as well as climate change have been listed as major reasons for a considerable increase in reported cases of foodborne parasitic zoonoses, leading to their recognition as an emerging public health problem. Therefore, prevention and protection against zoonotic parasites, especially in fishery products designated for human consumption, has recently become a priority. Consequently, health authorities and fishing industry have become aware of the significance of the nematodes of the genus Anisakis, affecting both human health and commercial value of fishery products. Although considered as one of the most significant emerging foodborne zoonosis, anisakiasis is still misdiagnosed and underreported in many Mediterranean countries. During a four-year research within the AnGEl project (Anisakis spp. Genomic Epidemiology) funded by the Croatian Science Foundation, we have assessed Anisakis nematodes using multidisciplinary approach, studying fish as paratenic, cetaceans as final and men as accidental hosts. The results of the project expanded our knowledge on: i) molecular geo-distribution of Anisakis spp. in the Adriatic fish inferred by a newly developed genome-derived small sequence repeats (SSR) panel, ii) histopathological effect of the nematode in the digestive tract of paratenic, final and model accidental host inferred by IHC and TEM, iii) nematode transcriptomic signature during migration through model host using RNAseq and microRNA profiling, iv) traits of the larval multixenobiotic resistance mechanisms that will help design more effective therapy, v) anti-Anisakis IgE seroprevalence in human population professionally related to fish processing industry. The most important results will be presented, aiming to trigger better conscience and take home message about this emerging and highly under- and miss-diagnosed zoonosis.

The aquaculture of the European sea bass Dicentrarchus labrax has been challenged by outbreaks of diseases, often occurring in intensive rearing systems. Increased administration of conventional chemotherapeutics can cause residue accumulation in marine environment and fish as final product and development of antimicrobial resistance, creating wrong image of aquaculture for the consumer. Probiotics, as an environmentally friendly prophylactic alternative, are beneficial and easy to use feed additives composed of live microorganisms that have an antimicrobial effect through modifying the intestinal microbiota, secreting antibacterial substances, competing with pathogens to prevent their adhesion to the intestine or for nutrients necessary for pathogen survival, eliciting an antitoxin effect and modulating the host immune system. It is proposed that bacteria of autochthonous origin have a greater capability to compete with resident pathogens and are also more prone to dominate and persist over other potentially pathogenic microbes. After random isolation of autochthonous gut bacteria of the European sea bass, their identification and pathogenicity testing, three potentially probiotic isolates were selected ; Pseudoalteromonas sp., Alteromonas sp., and Enterovibrio coralii. Selected isolates were tested and compared with a commercially available Lactobacillus casei isolate for their immunostimulative efficiency, inferring inflammatory, apoptotic and anti-pathogen response of sea bass’ peripheral blood leukocytes (PBLs). In order to evaluate early kinetics of the immune response, phagocytic activity, respiratory burst, and expression of lysozyme, Mx protein, caspase 3, TNF-α, IL-10 genes was measured 1, 3, 5, and 12 h post-stimulation by four bacterial isolates. Best immunostimulative properties were observed in Pseudoalteromonas- stimulated PBLs, followed by Alteromonas sp. and L. casei, while E. coralii failed to induce significant effect. In vitro assay results evidenced that intestinal autochthonous bacterial isolates have better immunostimulative properties on the host, compared to commercially available L. casei. A panel of biomarkers that represent pro-/anti-inflammatory, pro-/anti-apoptotic, and anti-bacteria/viral responses of the fish should be considered when evaluating the usefulness of the potential probiotic in aquaculture.

Anisakiasis, caused by accidental ingestion of live L3 stage larvae of Anisakis spp. through undercooked seafood, is a widespread, often underdiagnosed illness. One of the mechanisms commonly implicated in the development of drug resistance in nematodes is associated with the cellular efflux activity of ABC (ATP binding cassette) transmembrane transporters. Nematodes possess numerous homologues of ABCBs (Pgps and half-transporters) and ABCCs (MRPs) transporters with important physiological roles, aside from their role in multixenobiotic resistance. The scope of this study was to compare biocidal activity of two natural monoterpens (farnesol, nerolidol) and two synthetic widely-used anthelminthics (abamectin, levamisol) on Anisakis spp. larvae and the degree of involvement of ABC efflux mechanism in their pharmacokinetics. Ten Anisakis pegreffii larvae were exposed to growing concentrations of target chemicals ; experiment was conducted in six replicates with or without simultaneous administration of two inhibitors ; Valspodar (Pg-p inhibitor) and MK571 (MRP inhibitor). The concentration of target compound at which only 50% of larvae were alive (LC50) was determined: 135.9 μM for nerolidol, 134.4 μM farnesol, 0.3 μM abamectin and 0.84 for levamisol, significantly influenced by presence of one or both inhibitors for all chemicals except for abamectin. Toxicity curves were used to extrapolate doses where highest difference in larvicidal activity between the compound and one or both inhibitors was observed, suggesting it as point where ABC transporters elicit highest effect. Anisakis larvae were exposed to this concentration and sampled at 2, 6, 12 and 18 h after treatment for further analyses: quantification of Pg-p and MRP-like transcripts by real-time PCR, production of reactive oxygen species (ROS), intake of target compounds by HPLC, activity of transporters by Calcein-AM assay and effect on larval cytoskeleton by IHC and confocal microscopy. The results are discussed and provide integral view and understanding of the processes involved in larvicidal activity and protection mechanisms conferred by Anisakis pegreffii against tested compounds.

Microsatellites, also known as short sequence repeats (SSRs), are tandemly repeated nucleotide motifs (1-6 bp) found in nuclear genomes of many taxa. Great variations in the number of basic repeated units are characteristic for these loci, resulting in high heterozygosity and the presence of multiple alleles making them the markers of choice for various population and evolutionary genetic studies. We have used available genomic resources of Anisakis simplex hosted at WormBase ParaSite to conduct in silico screening for SSRs and develop first marker set for this nematode, the cause of zoonotic fish-borne anisakiasis. Genus Anisakis is comprised of nine species with low level of morphological differentiation at species level. It is generally assumed that A. simplex senusu stricto (s.s.) is more pathogenic to humans than other species, emphasizing the importance of correct species identification and possible occurrence of hybridisation between A. simplex s.s. and A. pegreffii in sympatric areas of parasitation. In total, 23, 574 microsatellites were identified in non-coding and coding regions, covering 0.31% of A. simplex genome inspected. When considering repeats from perfect and compound loci together, trinucleotides were most abundant followed by mono- and dinucleotides in all regions alike. Potentially informative non-coding loci were randomly selected, tested and optimised for multiplex PCR. Twenty-six primer-pairs gave a unique PCR product of expected size in A. simplex and A. pegreffii, three in A. ziphidarium and none in A. typica, reflecting their phylogenetic divergence. Twenty loci were further chosen to assess the relationship between individuals showing mito-nuclear signatures of both A. simplex and A. pegreffii, inferred through PCR-RFLP analyses of the ITS region and sequencing of the cytochrome oxidase 2 (cox2) mtDNA, in allopatric area of the Adriatic Sea. Using our panel of microsatellite loci, we inferred general admixture of all tested populations, except in North Sea-originating "pure" A. simplex s.s. Estimates of ancestry and interclass heterozygosity place most recombinant genotypes with parental class of A. pegreffii, indicating that observed mito-nuclear mosaicism is likely retention of ancestral polymorphism ; also questioning if pure A. simplex was ever recorded in the Adriatic Sea.

Consumption of raw or thermally lightly processed sea food contaminated by live third stage larvae of Anisakis spp. can lead to a zoonotic disease anisakiasis. In severe cases perforation of gastric or intestinal wall occurs caused by larval migration. Histopathologic changes in such cases include severe eosinophilia and subsequent formation of eosinophilic granulomas. The aim of the study was to compare two host models experimentally infected by Anisakis larvae ; Sprague-Dawley rat (Rattus norvgicus) as a model for human infection, and European sea bass, as a model paratenic host. Rats (n=15) and European sea bass (n=24) were each experimentally infected with 10 live third stage larvae of Anisakis spp. by oro-gastric intubation. Animals were euthanized, dissected and inspected for migrating larvae. Tissues with embedded larva migrans and consequent lesions caused by its migration were sampled and processed for classical histopathological analysis and transmission electron microscopy (TEM). In rats, Anisakis larvae were mostly found penetrating gastric wall and rarely intestinal wall, abdominal musculature or subcutaneous fat tissue. Histopathological analysis showed deep ulcerations of glandular epithelium in gastro-intestinal wall together with tissue necrosis at the site of larval penetration. Severe haemorrhages were present in lamina propria mucosae together with extensive neutrophil and eosinophil infiltration. In affected muscles structural disintegration and necrosis of muscle fibres was observed, accompanied by infiltration of neutrophils and eosinophils, some of which showed degranulation as revealed by TEM. In European sea bass, Anisakis larvae were found penetrating gastric or intestinal wall only in small number of infected animals indicating successful expulsion of nematodes by host's peristaltic. Lesions caused by larval migration were mostly characterized by ulcerations and necrosis of glandular epithelium and moderate infiltration of mast cells in lamina propria mucosae. Due to acute infection only, wound healing occurred rapidly and without proliferation of connective tissue. These findings demonstrate that rats, congruent to humans and both being accidental and naïve hosts, mobilize strong innate immunity resources against an unknown pathogen, such as Anisakis. In contrast, fish as an evolutionary long-lasting paratenic host exhibits considerably milder immune response with rapid wound healing processes.

Anisakiasis is an emerging fish-borne parasitic disease in humans caused by accidental ingestion of live third stage Anisakis sp. larvae. The aim of this study was to explore early infection mechanisms of Anisakis pegreffii larvae and the response of its accidental host, Sprague-Dawley rat, representing a mammalian model mimicking human infection. High-throughput next generation RNA sequencing was used to evaluate both in vivo experimental rat infection and in vitro, assessing transcriptoms of peripheral blood leukocytes (PBLs) stimulated with parasite extract. Seven rats were sampled at 6, 10, 18, 24 and 32h post-infection after being orally intubated with 10 A. pegreffii larvae (N=5) or 1.5 ml of NaCl (N=2). Total RNA was extracted from stomach and muscle tissues penetrated with Anisakis larvae, as well as the surrounding healthy tissue of infected rats (internal control) and non-infected external controls using TriReagent (Ambion, USA). RNA was also extracted from parasites found in different stages of infection ; non-migrating larvae in stomach and intestine, larvae in the process of stomach/intestine/muscle penetration and larvae freshly isolated from fish, as a control. In vitro effect of A. pegreffii protein extract was tested on rat PBLs, 1 and 12h post-stimulation, with non-treated PBLs as a control. Sequencing using Illumina NextSeq 500 of 16 pooled rat, 16 pooled A. pegreffii and 7 pooled PBLs samples resulted in approx. 30 million paired-end reads per sample, 75 bases long and of good average quality. Reads were filtered and mapped to Rattus norvegicus and A. simplex genomes. The host response is strongly directed towards inflammatory reaction, congruent with observed gross and histopathological findings, showing pathways of stimulated neutrophil chemotaxis, leukocyte migration, peptide secretion, as well as intense tissue remodelling marked by keratinocyte proliferation and apoptosis. De novo assembly of A. pegreffii transcriptome has shown a myriad of secretary and excretory products, previously noted as potent allergens. Gaining an improved understanding of development of anisakiasis in accidental host offers a possible pathway for discovering new methods of treatment and control of this disease.

Interactions between wild and farmed fish, in the context of parasite transmission between populations, remain poorly understood. Wild fish may be drawn into the vicinity of farms through the presence of abundant food, provision of shelter or by signals from conspecifics and can serve as reservoirs of parasite transmission. Similarly, cultured stocks may act to amplify infectious stages in the environment, as they may be present in high numbers, be more susceptible to disease than wild fish and may accumulate higher parasite burdens due to intense rearing conditions. The monogenean Sparicotyle chrysophrii and the cymothoid isopod Ceratothoa oestroides have been identified as the most devastating ectoparasites in Mediterranean aquaculture, inflicting losses primarily in gilt head sea bream Sparus aurata and European sea bass Dicentrarchus labrax production. Their life cycles and modes of transmission have not been completely elucidated and molecular resources for their investigation are extremely scarce. In order to address this, we have used a genotyping- by-sequencing approach, double digest Restriction-site Associated DNA sequencing (ddRAD-Seq), to generate a genome-wide SNP marker dataset with the long term aim of investigating the degree of differentiation among individuals collected from farmed and wild hosts. Initial trials were conducted with S. chrysophrii and C. oestroides samples collected from Croatian and Greek farms. Two separate ddRAD libraries for S. chrysophrii and C. oestroides were constructed (96 and 138 samples in total), producing over 16 and 21 million reads with 26, 566 and 95, 697 unique RAD loci identified respectively. Considerable variability in number of reads, polymorphic loci and SNPs identified was observed between individuals for both species. SNPs genotyped in at least 50% of the individuals were identified (507 in S. chrysophrii and 1, 364 in C. oestroides) and used to compare the structure between samples currently available: wild and farmed S. chrysophrii and F1 and F2 offspring propagated in the laboratory for C. oestroides. The results provide a baseline for development of new ecological and biological studies of these species, using new genomic tools and resources, with important implications for aquaculture management and practice.

Consumption of raw or undercooked sea food infected with live third stage larvae (L3) of the nematode Anisakis spp. can lead to a zoonotic disease termed anisakiasis or anisakidosis. Although symptoms can vary greatly, in severe cases of disease perforation of gastric or intestinal wall, due to migration of the larva, occurs. Histopathologic changes in such cases typically exhibit severe eosinophilia and formation of eosinophilic granulomas as hallmarks of gastro-intestinal anisakiasis in humans. The aim of this study was to compare two host models experimentally infected with Anisakis larvae, namely Sprague-Dawley rat (Rattus norvegicus) as a model for human infection, and European sea bass (Dicentrarchus labrax) as a model paratenic host. Rats (n=15) and European sea bass (n=24) were each experimentally infected with 10 live third stage Anisakis larvae by oro-gastric intubation. At predefined time points, animals were euthanized with an overdose of anaesthetic, dissected and inspected for migrating larvae. Tissues with embedded migrating larva and lesions caused by its migration were sampled and routinely processed for classical histopathological analysis and transmission electron microscopy (TEM). In rats, larvae were mostly found penetrating gastric wall. Only rarely, larval migration through other tissues, i.e. intestinal wall, abdominal musculature or subcutaneous fat tissue, occurred. Histopathological analysis showed deep ulcerations of glandular epithelium in gastro-intestinal wall accompanied by tissue necrosis at the site of larval penetration. In lamina propria mucosae severe haemorrhages with extensive neutrophil and eosinophil infiltration were present. Affected muscles were infiltrated with neutrophils and eosinophils, some of which showed degranulation as revealed by TEM. Moreover, necrosis and structural disintegration of muscle fibres was observed. In European sea bass, Anisakis larvae were found penetrating either gastric or intestinal wall in a few animals only, indicating successful expulsion of parasites by host’s peristaltic. Larval migration through gastro-intestinal wall caused ulcerations and necrosis of glandular epithelium. Lamina propria mucosae was moderately infiltrated with mast cells that tended to accumulate around larvae themselves. Due to acute infection only, wounds caused by larva migrans healed rapidly and without proliferation of connective tissue. These findings demonstrate that rats, identical to humans as both being accidental and naïve hosts, exhibit strong innate immune response to an unknown pathogen, such as Anisakis. In contrast, fish as an evolutionary long-lasting paratenic host shows considerably milder immune response to Anisakis parasite together with rapid wound healing processes.

Atlantska plavoperajna tuna Thunnus thynnus je pelagična riba iz porodice Scombridae, koja nastanjuje Atlantski ocean i Sredozemno more duž kojih migrira u svrhu reprodukcije i ishrane. Zbog svog hranjivog i ukusnog mesa iznimno je cijenjen ulov, pogotovo u Japanu, što je potaklo razvoj uzgoja ove vrste u Sredozemlju, Australiji, dalekom Istoku i Americi. Od 1996. godine prakticira se njen kavezni uzgoj u Hrvatskoj gdje čini veliki dio izvoza ribarstvene industrije i donosi značajnu ekonomsku dobit. Ulov, transport, adaptacija na kavezni uzgoj, loša prehrana, nagle promjene okolišnih čimbenika uzrokuju stresne uvjete u ribe što može rezultirati utišavanjem komponenti urođene imunosti. To dovodi do osjetljivosti na infekcije i izloženosti patogenima što loše utječe na rast i kondiciju i može u konačnici uzrokovati mortalitet juvenilne ribe i velike gubitke u akvakulturi. Mikrosateliti su najčešći oblik ponavljajuće DNA u eukariotskom genomu, lako se amplificiraju PCR reakcijom i predstavljaju odlične genetičke biljege u istraživanju populacijske strukture, evolucije, identifikacije jedinki, određivanju srodstva među jedinkama te se često koriste u genetičkim i medicinskim istraživanjima. Koristeći mikrosatelitne motive identificirane na sekvencama normalizirane cDNA knjižnice plavoperajne tune kao inicijalnog kalupa, razvijen je multipleks PCR protokol s 14 lokusa smještenih u blizini kodirajućih regija gena koji se eksprimiraju prilikom stresa i imunosne reakcije. Na uzorcima DNA izoliranim iz fragmenta peraja 334 tune uzorkovanih prilikom ulaza populacije u uzgoj, te prilikom izlova iste populacije nakon 1.5 godišnjeg uzgoja utvrđena je razlika u učestalosti polimorfizama ovih mikrosatelitnih lokusa, što se može dovesti u vezu s fitnessom jedinki u uzgoju. Istraživani lokusi u budućnosti mogu poslužiti kao biljezi za otkrivanje jedinki unutar populacije koje su uslijed polimorfizama unutar imunosnih gena sklonije promjenama imunosnog sustava i imaju izraženiji rizik od ugibanja kroz uzgojni ciklus.