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Trabajo presentado en el 6th International jellyfish blooms symposium, celebrado en Cape Town del 4 al 6 de noviembre de 2019., The symbiotic association between the zooxanthellae Symbiodinium sp. and its host jellyfish, the rhizostome scyphozoan Cotylorhiza tuberculata was studied. Measurements were carried out in two different sections of the medusa body to investigate de distribution of the alga in hospite: oral arms (OA) and umbrella (UM). Medusa carbon and nitrogen composition were higher in the OA section than UM (4.43 ± 0.3 and 3.7 ± 0.26 respectively). No significant differences were found in the chlorophyll a content among the two sections. However, zooxanthellae density was higher in OA than UM tissue. A total of 13 different pigments was revealed by the High Performance Liquid Chromatography (HPLC) method, the most representative pigments were chlorophyll c2 peridin and diadinoxanthin, typical pigments of dinoflagellates. Cell diameter was significantly higher in hospite (8.71 ± 0.94 μm) than in culture (6.78 ± 0.85 μm), whose growth rate value was 0.285 (± 0.09) d-1. Cultures of Symbiodinium sp. observed by light microscopy, cytograms, and SEM micrographs, showed three subpopulations of autotrophic cells: a bigger and a smaller coccoid stage, and a motile flagellate stage. The presence of zooxanthellae living in symbiosis with C. tuberculata may be a reason added to explain the population success of this jellyfish.

The risk posed by complex chemical mixtures in the environment to wildlife and humans is increasingly debated, but has been rarely tested under environmentally relevant scenarios. To address this issue, two mixtures of 14 or 19 substances of concern (pesticides, pharmaceuticals, heavy metals, polyaromatic hydrocarbons, a surfactant and a plasticizer), each present at its safety limit concentration imposed by the European legislation, were prepared and tested for their toxic effects. The effects of the mixtures were assessed in 35 bioassays, based on eleven organisms representing different trophic levels. A consortium of 16 laboratories was involved in performing the bioassays. The mixtures elicited quantifiable toxic effects on some of the test systems employed, including i) changes in marine microbial composition, ii) microalgae toxicity iii) immobilization in the crustacean Daphnia magna, iii) fish embryo toxicity, iv) impaired frog embryo development and v) increased expression on oxidative stress-linked reporter genes. Estrogenic activity close to regulatory safety limit concentrations was uncovered by receptor-binding assays. The results highlight the need of precautionary actions on the assessment of chemical mixtures even in cases where individual toxicants are present at seemingly harmless concentrations., JRC.H.1-Water Resources

Photosynthetic pigments have proved to be useful biomarkers of the abundance, composition and physiological status of the phytoplankton biomass in the marine environment. Using HPLC pigment analysis, we determined phytoplankton community structure in three different marine environments: in the area of a fish farm, in the area of sewage outlets, and in the mucilaginous aggregates. At the reference site we observed seasonal changes with prevalence of fucoxanthin-containing phytoplankton (i.e. diatoms) during winter/spring and autumn. In the fish farm area the concentration of chlorophyll a degradation products was higher, whereas in the locally enriched environment of sewage outlets we observed only small changes in taxonomic composition and phytoplankton biomass. The impact of season is more expressed than the impact of sewage discharge. With the use of HPLC pigment analysis we determined the development of phytoplankton community in different stages of mucilage aggregates. Phytoplankton biomass was composed primarily of diatoms, and as the aggregates aged, diatoms increased in the relative biomass. Our examples have proven the usefulness and suitability of HPLC pigment analysis in marine ecology studies.

Vertical distribution, population structure and chemical composition of a population of the schyphomedusan Aurelia sp. 5 wewre studied in the Mediterranean eurohaline lake on Mljet Island (Veliko jezero, the Southern Adriatic) where dense aggregations have been observed throughout the year. A majority of Aurelia was located in layer below the thermocline with temperature 13-19 0C over the most of the day during summer, whereas in October the population was shallower. The medusae migrated toward the surface at dusk but rarely crossed the thermocline. Elemental analysis of Aurelia revealed low dry specific carbon, nitrogen and phosphorus content ranging from 0.9-1.7%, 0.2-0.5%, amd 0.02-0.06% respectively. In addition to the direct predation impact on micro- and mesozooplankton, a clear positive response of heterotrophic bacteria to the presence of live and dead Aurelia was observed.

The occasional extensive appearance of mucous aggregates in the summer period is specific to the northern Adriatic. We followed this phenomenon from July to September 1997, focusing on temporal changes of phytoplankton in mucous aggregates as reflected by biomarker pigments. The qualitative and quantitative analyses of pigments (chlorophyll and carotenoid pigments as well as breakdown products of chlorophyll a) were determined using reversed-phase high-performance liquid chromatography (RP HPLC) equipped with serially-coupled spectrophotometric and spectrofluorimetric detectors. The phytoplankton biomass in mucilage samples, as expressed in chlorophyll a concentrations, was very high and ranged from 19.9ą7.2 to 141.5ą32.4 microg/g dry mucilage. The largest percentage of the autotrophic biomass in all samples was associated with fucoxanthin-containing phytoplankton i.e. diatoms (from 21.0ą3.9 to 148.6ą34.4 microg fucoxanthin/g dry mucilage) that contributed from 40.8ą2.1 to 74.7ą1.6 % of the total biomass, followed by peridinin (dinoflagellates), zeaxanthin (cyanobacteria), chlorophyll b (green algae), 19'-hexanoyloxyfucoxanthin (prymnesiophytes) and 19'-butanoyloxyfucoxanthin (silicoflagellates). Contribution of those individual biomarkers was generally less than 15 %, except for peridinin-containing species, which reached nearly 40 % in August. These results suggest that microplankton, especially diatoms, play a key role in the development and maintenance of mucilage. The total concentration of breakdown products of chlorophyll a was found in the range from 750ą160 to 19330ą109 ng/g dry mucilage. The most abundant breakdown product was chlorophyllide a, followed by phaeophorbide a_1, phaeophorbide a_2, phaeophytin a_1 and phaeophytin a_2.

The occasional extensive appearance of mucus aggregatesnin the summer period is specific to the northern Adriatic. We followed this phenomenon from July to September 1997 focusing on temporal changes of phytoplankton in mucous aggregates as reflected by biomarker pigments. The largest percentage of the autotrophic biomass in all samples was associated with fucoxanthin-containing phytoplankton (21-144 microg/g dry mucilage) which contributed 41-75 percent of the total biomass. Contribution of other individual biomarkers was generally less than 15 percent, except for peridinin-containing species, which reached nearly 40 percent in August. These results suggest that microplankton, especially diatoms, play a key role in the development and maintenance of mucilage. The composition of phytoplankton biomass in the surrounding waters was markedly different. In the surface layer (less of 10m) the unusual prevalence of fucoxanthin-containing biomass in July was followed by a more typical system dominated by pico-/nanoplankton in August. In contast, the lower laxer (more than 10m) was strongly dominated by microplankton (fucoxanthin and peridinin) troughout the summer. These data indicate a temporary decoupling of phytoplankton dynamics in mucilage and surrounding water

The dry matter and ash-free dry matter (DW, AFDW), the particulate organic carbon (POC), and the particulate organic nitrogen (PON) content of suspended mucous aggregates and seston were determined in the Gulf of Trieste during the 1997 (July-August) mucilage event. Seston was sampled with 5L Niskin bottles while mucous aggregates were collected by SCUBA divers using a peristaltic mucous aggregates was estimated on the basis of in situ SCUBA diver observertions, photography, and video-recording of vertical profiles of their sizes, types and abundances. Organic seston (AFDW) concentration varied from 1.2 to 2.6 mg/L, which was within the normal range for the summer period. The concentration of organic matter associated with mucious aggregates (0.2 to 6.1 mg/L) contributed significantly to the total suspended organic matter indicating that a significant part of primary production was channeled into mucilage. It was estimated that during 2 mounth period about 40 percent of the total primary production accumulated as C-rich (C/N ratio 9.8-18.0) mucilage organic matter.

During two cruises of the MEDUZA experiment conducted in July 2003 and May 2005 various data were obtained over the deepest part of the southern Adriatic Pit. Temperature, salinity, light transmission and fluorescence were measured in July 2003. During May 2005, except CTD measure- ments the samples were taken for laboratory determination of phytoplankton pigments with HPLC technique. During both cruises irradiance and radiance were measured at 14 wavelengths in the range 340-715 nm with optical probe to about 100 m depth. Bad weather conditions during both cruises caused interruption of measurements that continued after 24 to 48 hours. Stormy conditions have resulted in a slightly different structure of the water column. In the periods of measurements recorded were diurnal rhythms in a series of parameters, caused by solar radiation cycles in the surface layers as well as fluctuations in the deeper layers under the influence of the internal tide. Meteorological conditions and changes in sea level are also described. The analysis included the principal component and correlation analysis between the measured and derived parameters in order to establish a link between the in-situ pigments and multispectral data. During both cruises diurnal vertical oscillations were observed in biotic and abiotic parameters. Chlorophyll pigments prevailed over other pigments preventing other pigments to be distinguished spectrally. Regression models for chlorophyll a were established from radiance and reflectance ratios.

Fish communities within two Croatian MPAs were surveyed using both diver-operated underwater video (DOV) via SCUBA and lure-assisted visual census (Lure-UVC) via snorkling. Both methods were applied at similar depths within replicate MPA locations, Kornati NP (4) and Brijuni NP (2). We predicted that the methods detect similar fish communities, habitat mosaics, and fish habitat preferences. To avoid inter-observer variability- errors, fish and habitat identification in all surveys was done by the CK. Habitat types were defined by a common protocol. Higher species richness (35 > 30) was detected by lure-UVC in Kornati. In Brijuni, DOV detected more species (31 > 25). The MPA-specific communities observed by the two methods were 72% similar within Kornati NP and 92% similar within Brijuni NP (Sørensen Index). Both methods recognized the same six habitats in Brijuni NP and five common habitats in Kornati NP, with one unique observed by DOV. In Kornati NP, Lure-UVC detected significant habitat preferences for 10 (28%) species of which nine preferred transitional habitats, the edges between R and U (RU) and R and P (RP) ; DOV detected significant habitat preferences for 15 (50%) species of which 12 preferred either R or RP transitions. In Brijuni NP lure-VC detected four (13 %) and DOV seven (28 %) species with significant habitat preference, and all eleven species were significantly attracted to transitions from rocky reefs to sedimentary bottoms (RU) and all significantly avoided bare sand (U). We conclude that the two methods are comparable in their ability to sample fish communities, habitats and habitat preferences. Decisions about which method to use should focus on availability of resources and target depths. Lure-UVC is more cost and time efficient because it does not need SCUBA and video-processing but requires the presence of a fish/habitat expert in the field, preferably the same individual. DOV requires costly SCUBA operation and lengthy post- video analysis in the lab but allows for multiple experts relying on permanent records. Sample sizes per unit field-time is high for DOV and depends on the number of divers operating DOV. DOV can be applied at wider depth range than surface-based UVC.

Doliopsis rubescens was a very common species in the Mediterranean in 19 th and in the beginning of the 20 th century. In the past it was a very abundant species, so much that it formed large blooms at the coast of France. Afterwards it has become extremely rare with sporadic records in the Atlantic. D. rubescens was found in the Adriatic for the first time in 2016 and 2017, in the depth layer from 50 to 200 m. Two individuals preserved in ethanol were sequenced and the phylogenetic tree was constructed based on 18S rRNA gene sequence. The phylogenetic position of D. rubescens is discussed in relation to other Thaliacea and its morphological peculiarities.

Detection of parasites and pathogens of molluscs allows the management of natural stocks and disease control in native and cultured species. In this study, we investigated digestive gland cryosection of the Mediterranean mussel Mytilus galloprovincialis Lamarck, 1819 sampled at two locations (St. Andrew and ACI Rovinj) in the northern Adriatic Sea across annual cycle. The protozoan of the genus Nematopsis Schneider, 1892 (Apicomplexa, Gregarina) have been found for the first time in cryosections of mussel from both sampling stations. One to three oocyst of Nematopsis sp. were detected in most cryosections located within phagocyte in connective tissue between digestive tubules of mussel digestive gland. In some cryosections, five to eight oocyst were observed. The prevalence of Nematopsis sp. ranged from 20% to 100% in the one-year study. Intensity of infection was variable, with < 30 oocyst per section in most of the cases (maximum 110 oocysts/mussel). We observed the parasite infection in relation to habitat, time of sampling and environmental factors.

Fish stocks should be monitored with fisheries-independent and non-destructive methods. In Croatia, we are using baited, remote, underwater, stereo video (BRUV) and diver-operated videovisual census (DOV), potentially operated inexpensively by personnel untrained in fish biology, but requiring lengthy and tedious office labor by experts to classify, count, and measure fish. Automated methods will potentially greatly reduce this labor cost and allow greater video dataprocessing per unit time, and increased statistical power for detecting spatio-temporal variation in fish populations and fish communities. We are currently developing computer-vision tools for automated processing of high resolution underwater videos from BRUV and DOV videos taken in the shallow Croatian Adriatic under varied fish assemblage, water, and habitat conditions. The method is based on deep machine learning and consists of three major steps: detection, classification, and movement tracking of fish. The current prototype achieves accuracies for fishspecies classification of 69%, 94% and 98% on the Croatian dataset (794 images), the fish4knowledge 2012 dataset (27370 images) and the seaclef 2015 dataset (22443 images) respectively. The positive correlation between dataset volume and classification accuracy indicates that experts should annotate over 25000 images for the algorithm to be trained to achieve accuracies above 90%. In the near future we plan to embed our algorithms into a lifelong machinelearning framework that continuously improves through incremental learning from newly arriving annotated data. Human-machine collaboration is facilitated by the software tool L3P which allows for annotation within a web-based image-processing engine. This tool enables fish experts to improve analysis algorithms in the cloud without specialized computer-vision knowledge. Our core system achieved best results in a competition to estimate fish abundances within the seaclef 2016 dataset and is foreseen to accomplish several more specialized tasks. We present an innovative machine learning approach that will utilize human-machine collaboration and is suitable for the overall challenge of monitoring marine species and habitats. This work was partially supported by the Croatian Science Foundation, under the project COREBIO (3107).

Lure-assisted visual census, a fisheries-independent and non-destructive method for quantifying fish abundance and benthic habitat, was used to assess fish communities inside Croatian (eastern Adriatic Sea) marine protected areas, and outside these areas at similar locations. Marine protected areas included National Parks Brijuni, Kornati, Mljet, and Nature Park Lastovo. A total of 19678 visual transects were followed at 149 locations throughout the Croatian nearshore at depths from two to seven meters. Relative species abundances were compared within a generalized linear mixed model with MPA protection as a fixed effect and location as a random blocking effect, and species accumulation curves were constructed using Chao, jacknife, and bootstrapping methods. We found the total species richness was slightly higher inside all four MPAs than outside, but communities were highly significantly different inside versus outside all MPAs. The results indicate that organization of fish communities is highly sensitive to 1) regulation of fishing activities, and 2) degradation of bottom habitat, such that commercially valuable predatory species (including Diplodus sargus, D. puntazzo, Mullus surmuletus), and species dependent on intact and undisturbed macro-vegetation (Oblada melanura, Sarpa salpa) and rocky reef habitat (Epinephelus marginatus) are more abundant inside MPAs where habitat is less disturbed. Lure-assisted visual census is a powerful non-destructive method for quantifying the effects of fishing regulations on populations of common and commercially important fish species. This work was partially supported by the Croatian Science Foundation, under the project COREBIO (3107).

Baited remote underwater video is a non-destructive method for censusing fish species abundance that is currently under consideration for use in the Mediterranean Sea for monitoring fish stocks and the effectiveness of fisheries regulations. We tested the method in the eastern Adriatic for effectiveness in detecting impacts of habitat degradation and fish harvest on fish abundance, size, and community assembly. A total of 215 BRUVs were deployed at 10 locations during all months from April to September of 2015 and 2016 at and near Kornati National Park (KNP) in Croatia, at benthic habitats including unconsolidated sand/gravel to rocky reefs, with and without macro vegetation consisting of seagrasses Posidonia oceanica, Cymodocea nodosa, or macroalgae Cystoseira species. We analyzed species abundance using generalized linear models with protection or habitat status as fixed predictor factors, location and date of sampling as a random blocking factor, and the maximum number of individuals visible within any single video frame as the proxy of abundance within a species. To estimate asymptotic total species richness we used the Chao estimator, or standard methods of jacknifing or bootstrapping the dataset. We found very highly significant differences in species richness across 10 sampling locations, with the maximum estimated richness within KNP ranging from 53 to 65 (SE 2 to 10), and outside KNP ranging from 24 to 27 (SE 1 to 2). In addition, we find significantly higher species richness associated with structured and edge habitats, such as rocky reefs, and lower richness associated with uniform seagrass or anthropologically degraded habitat. Taxa found more abundant inside the MPA include species heavily targeted by local fisheries, such as Diplodus species, Spaurus aurata, Dentex dentex, Mullus surmuletus, and schooling pelagic genera such as Spicara. We conclude that BRUV has power to detect responses of individual species both to harvest, and to human habitat disturbance, and can assist in testing hypotheses about fish community assembly in the Adriatic Sea. This work was supported by the Croatian Science Foundation under the project COREBIO (3107).

The Adriatic Sea is a semi-enclosed sea and its ecosystem is influenced by the regular exchange of water with the Eastern Mediterranean. Levantine Intermediate Water and Ionian Surface Water flow into the Adriatic along the sea’s eastern border. The volume of this flow depends on climatic oscillations that occur from the Atlantic to the Southeast Mediterranean. The South Adriatic, the deepest part of the Adriatic Sea, represents a key area for both the Adriatic Sea itself and the entire Eastern Mediterranean basin. Tintinnids are the best known ciliates in the marine plankton. Their high frequencies and identifiable morphology have been suggested as indicators of oceanographic conditions including water temperature and origin, such as neritic, oceanic, and even upwelling water. Samples in South Adriatic were collected at four stations with a 53 µm mesh Nansen net. The spatial and temporal distribution of tintinnids was investigated at monthly intervals in the South Adriatic Sea during 2016. Of the 68 species identified, there were 47 surface, 15 subsurface, 3 mid-water, and 1 deep sea species. The dominant surface and subsurface species were Codonella aspera, Codonella amphorella, Undella claparedei and Salpingella accuminata. Species Parundella lohmanni predominated in the mid-water layers and Xystonellopsis scyphium in the deep-sea layers. While the majority species are always present in the South Adriatic Sea, some species were occasionally immigrates from the Mediterranean Sea. The highest variability in the number of species occurred in the surface and subsurface layers. A mid-water community of species Parundella lohmanii indicated inflowing South Adriatic Deep Water. Some other species such as Poroceus apiculatus, Undella subcaudata and Eutintinnus elegans appeared with strong inflows of the Levantine Intermediate Water during winter and spring. Based on these results, tintinnids may be used as a biological indicator of the state or possible changes in the marine ecosystems of the South Adriatic Sea.

The Indo-Pacific red alga Lophocladia lallemandii (Montagne) F. Schmitz 1893, has been probably introduced in the Mediterranean Sea through the Suez Canal. It was first recorded in Greece and Lybia in 1908, and since then, over a wide area of the Mediterranean Sea. In some locations, L. lallemandii shows invasive behavior with negative consequences on Posidonia oceanica meadows, native macroalgae and marine invertebrate. It is considered one of the 100 most dangerous alien Mediterranean marine species. In the Adriatic Sea it was found only in few locations: at the entrance to the Adriatic Sea - Sazani Island (Albania), Tremiti Islands (Italy) and the Gulf of Trieste (Italian and Slovenian side). According to recent information, in the northern Adriatic is not present anymore. Here we report a new location for the Adriatic Sea which we discovered in October 2012 on Blitvenica Island (Middle Adriatic). It was found as an extensively developed species. This represents the first record in the East part of the Middle Adriatic which is also one of the northern locations in Mediterranean. Since the first observation, L. lallemandii was constantly present with full development in autumn. Seawater bottom temperature at 15 m measured by data loggers from October 2014 till October 2016 has average values of 18.2 C°, with the minimum 12.8 C° in March 2015 and maximum 26.1 C° in July and August 2016. Species develops between 1 and 30 m, mainly as an epiphyte on Cystoseira and Sargassum species. In October 2016, the maximum coverage of 12% between 5 and 15 m depth was found. Reproductive structures were noted: trichoblasts and slightly spirally twisted tetrasporangial stichidias occurred on basal cells of trichoblasts. Based on previous studies on invasive behavior of this species in other parts of the Mediterranean and extensive development on Blitvenica Island, we believe that this species could negatively affect the special composition of the communities of the region where some very rare species exist, such as Cystoseira jabukae. Also, there is a possibility of further spreading across the Adriatic Sea, where it could have an impact on the native benthic biota and habitats.

The northern Adriatic is characterized by particularly steep ecological gradients over space and time. Some of those gradients are strongly connected to benthic pelagic coupling, like e.g. the sudden availability of nutrients during the onset of water column mixing. Living species have to adapt to abiotic processes in the ecosystem and of course to processes in benthic pelagic coupling. However, they also play an active role in the mechanisms driving benthic pelagic coupling. The analysis of aggregates in the water column and the metabolic activity of planktonic species in and around those aggregates exemplifies the danger of generalization, black box approaches and again the need for in situ observations. It also demonstrates how the borders between benthic and pelagic life can be crossed in both directions. To understand adaptations to and active roles in such processes, meticulous in situ observations are of pivotal importance. The large variability in phytoplankton species (and other pelagic species) stands in strong contrast to the apparent continuity and lack of structure of their environment. However, research on phytoplankton life strategies, in particular with regard to competition for nutrients, unveils a rather rich structure of the pelagic ecosystem and a correspondingly rich variability of life strategies. Such rich variability can be found for interactions between different planktonic organisms as well. Food preference as well as grazing efficiency of planktonic and benthic species vary throughout their lifecycle already and showcase the complexity of the biology behind benthic pelagic coupling.