Lim Bay Research Articles

Performance of mussel Mytilus galloprovincialis under variable environmental conditions and anthropogenic pressure: A survey of two distinct farming sites in the Adriatic Sea

Changes in natural conditions and anthropogenic pollutants, alone or in combination, pose a significant challenge to coastal bivalve populations. The susceptibility of economically important bivalves to potential stressors in their farming environment has not been sufficiently investigated, despite the increase in anthropogenic pressure along the coast and the remarkable warming of seawater in recent years. Thus, the aim of this study was to evaluate the performance of mussel (Mytilus galloprovincialis) from two important farming sites in the eastern Adriatic, namely Mali Ston Bay (MSB) and Lim Bay (LB), in relation to variations of seawater parameters, reproductive cycle dynamics and tissue content of potentially harmful pollutants. The complex seasonal and site-specific patterns of chemical pollutants were determined, with tissue levels of metals, As, PAHs and PCBs largely comparable to those previously reported for the Mediterranean region. Concentrations of organochlorinated pesticides were below the level of detection. Significantly higher Cd, As and Hg concentrations were detected in the tissues of the MSB mussels. The reproductive cycle was clearly associated with the bioaccumulation of pollutants. All biochemical response parameters varied to some extent across seasons and/or between farming sites. A very pronounced seasonality was recorded for acetylcholinesterase and glutathione S-transferase activity at both sites. Metallothionein concentration and superoxide dismutase activity were generally steady throughout the study period. The most striking difference between the two sites was recorded for lipid peroxides concentrations which were predominantly significantly higher in the MSB mussels, indicating expressed pro-oxidant conditions at this site. In particular, significant correlations were found between lipid peroxides and the potentially toxic metals (Cd, As, Hg) accumulated in the mussel tissue. Data reported here are valuable as baseline information for further studies related to stress in farmed bivalves caused by oscillations of environmental factors and increasing anthropogenic pressure along the coastline.

Diversity, virulence and antibiotic resistance of Vibrio Harveyi clade species associated with bivalve aquaculture within marine protected areas

The aim of the present study was to investigate the diversity, distribution of virulence factor genes and antimicrobial resistance phenotypes of Vibrio Harveyi clade species from two distinct and spatially distant near-shore bivalve farming environments located in protected marine reserves of the eastern Adriatic. Tissues of farmed Mediterranean mussel Mytilus galloprovincialis and European flat oyster Ostrea edulis, seawater and sediment samples were collected during six bi-monthly sampling events conducted over one year from farming locations in Lim Bay and Mali Ston Bay, Croatia. Eight distinctive Harveyi subclades, namely, V. harveyi, V. alginolyticus, V. diabolicus, V. jasicida, V. parahaemolyticus, V. rotiferianus, V. campbellii and V. communis, were detected by Phylogenetic Multilocus Sequence Analysis (MLSA) based on rpoD, toxR and rctB concatenated genes sequences. Genomic fingerprinting of MLSA-classified samples revealed substantial diversity of environmental isolates. The virulence-related genes considered typical for the Harveyi clade and their homologues were represented in all subclades with varying frequencies. Of twelve antibiotics commonly used in human and veterinary medicine, the most frequent resistance phenotypes were resistance to ampicillin (90%), erythromycin (39%), imipenem (29%) and streptomycin (22%). >60% of isolates were resistant to at least two classes of antibiotics. Seasonal or site-specific differences were found in the proportion of isolates carrying virulence genes and displaying specific antibiotic resistance phenotype.This study is the first to provide a broader perspective on the properties of Vibrio Harveyi clade species associated with ecosystems under presumably low anthropogenic pressures, such as marine protected areas. New data on virulence and antibiotic resistance are presented of Harveyi clade species coexisting in different environmental compartments, including those that have been largely overlooked or understudied in the context of bivalve aquaculture. Taken together, these results highlight the importance of monitoring the presence of potentially pathogenic strains in the natural Vibrio population of bivalve farming areas to reduce the risk to seafood consumers.

Competitive feeding interactions between native Ostrea edulis and non-native Crassostrea gigas with implications of introducing C. gigas into commercial aquaculture in the eastern Adriatic Sea

In order to detect the possible regulatory effect of non-native C. gigas on the native O. edulis, under aquaculture conditions, feeding interactions between them were investigated in a highly productive environment of Lim Bay (Adriatic Sea). The present study uses a multi-methodological approach, including stomach content, DNA barcoding and stable isotope analysis to elucidate the feeding ecology of two oyster species. The research confirmed a high overlap throughout the year in the feeding traits among native and non-native oyster species. Competition for food was not the only relationship that exists between the investigated species as the presence of O. edulis larvae in C. gigas stomach content was confirmed by DNA analysis. Findings are not in favour of introducing C. gigas to commercial aquaculture in any new areas in the Adriatic Sea and support the need to improve the existing O. edulis aquaculture and conserve its wild stocks.

Reproductive cycle of a non-native oyster, Crassostrea gigas, in the Adriatic Sea

The Pacific oyster Crassostrea gigas was introduced for the aquaculture purposes in many different parts of the world. However, the species has never been officially introduced for commercial farming in the Croatian part of the Adriatic Sea. Interestingly, in the 1970s, the Pacific oyster has been reported in the natural habitats of the Lim Bay, in northern Adriatic Sea. Although the species was recorded there, there is a lack of knowledge about its biology and ecology in this region, including a reproductive cycle. Information on the reproductive biology of non-indigenous species in new areas is crucial for prediction of their future survival and possible spread in invaded habitats. In this study, we provide the first data on the reproductive biology of the Pacific oyster in the Adriatic Sea, the northernmost part of the Mediterranean Sea. Quantitative and qualitative methods of gonad tissue analysis were conducted, and effects of environmental conditions on the gametogenesis were evaluated during two reproductive cycles. Our study confirmed that environmental conditions in the Lim Bay were favourable for the reproduction of the species. The Pacific oyster reproduced once per year and had a prolonged spawning period. Water temperature was the main factor affecting gonad development and oocyte size, while chlorophyll a concentration had an impact on the oocyte development in terms of their size.

Distribution patterns of two co-existing oyster species in the northern Adriatic Sea: The native European flat oyster Ostrea edulis and the non-native Pacific oyster Magallana gigas

Abstract The Pacific oyster Magallana gigas, globally one of the most translocated marine species, has never been commercially farmed in any part of the Croatian eastern Adriatic Sea, where the native flat oyster Ostrea edulis is the only cultured oyster species. The Pacific oyster has, however, established populations on the west coast of the Istria peninsula, in the northeast Adriatic Sea. Current distribution, abundance and size structure of the flat and Pacific oysters were studied there along spatial and depth gradients in natural and artificial habitats to assess their potential for coexistence. This is the first quantitative assessment of native and non-native oyster populations on a Mediterranean-wide scale providing a baseline for determining future changes in their distribution. Both species were omnipresent, with the Pacific oyster displaying a more pronounced variability in abundance and size in relation to survey regions and depths. The population density of flat oysters was low, generally less than 1 individual/m2, with no difference among regions. The density of the Pacific oyster was significantly higher, being on average 5 individuals/m2. Dense, reef-forming aggregations of Pacific oysters were contained in Lim Bay, a nationally-important shellfish aquaculture area, where its mean density was 107 individuals/m2. Along the open coastline its densities were considerably lower and followed a latitudinal gradient. The observed abundance and size distribution patterns of the Pacific oyster suggest that Lim Bay was the introduction point, with feral settlement likely originating from short-lived experimental aquaculture trials and subsequently dispersing by prevailing local currents. The flat oysters were larger in size and settled at different depths compared to the Pacific oysters. The vertical range of Pacific oysters was mostly contained in the tidal zone, while flat oysters were present in the subtidal exclusively. Such spatial partitioning likely resulted from introduced oysters occupying a vacant ecological niche and not due to interspecific competitive exclusion. Habitat type had a strong effect on proliferation of Pacific oysters. Artificial hard substrata harboured more abundant and larger Pacific oysters than the natural rocky shore habitat. The possibility of multiple local introduction pathways in ports and marinas is also discussed. Currently, there seems to be no spatial competition between the two oyster species, but as it is hard to predict future possible impacts of non-native species, we strongly suggest regular monitoring of the Pacific oyster, prioritising sensitive and protected areas as well as areas at the edge of its distribution.

Rasprostranjenost nezavičajne vrste kamenice Magallana gigas(Thunberg, 1793) duž istočne obale Jadrana

Non-native Pacific oyster Magallana gigas (Thunberg, 1793) was introduced to the Mediter-ranean Sea for aquaculture purposes in the 1960s. Although this species was not introduced for aquaculture to the Croatian part of the Adriatic Sea, in the 1970s, it was reported in the Lim Bay, in the North-eastern Adriatic. Until recently, there has been no research on the species in the Croatian part of the Adriatic. The aim of this research was to summarize existing and novel data on the distribution of M. gigas in coastal areas of the Eastern Adriatic and to provide a baseline for the future monitoring and assessment programmes of the species. Distribution of M. gigas was determined by three different methods: (i) a visual census of the presence of M. gigas specimens in the medio-littoral zone; (ii) DNA identification of M. gigas larvae in the water column; and (iii) the presence of M. gigas in the subtidal zone at depth between 25 and 40 m. Magallana gigas has a well-established population in the medio-littoral zone of natural and anthropogenic habitats along the coast of the North-eastern Adriatic Sea (west coast of Istria peninsula), but it is not present in the deeper layers . In the Central-eastern and South-eastern Adriatic Sea, the species was either absent or sporadically recorded with no evidence of fully established populations. Considering the great invasion success of M. gigas worldwide and effects that this species could have on the invaded ecosystem (e.g. competition for food and space with native species), detailed future monitoring is needed for the Eastern Adriatic Sea.

Adaptation of cultured mussel Mytilus galloprovincialis Lamarck, 1819 from the northern Adriatic Sea to nearby aquaculture sites and translocation

The ability of mussels to cope with environmental conditions depends on the species’ capacity to acclimate or adapt within its morphometric and physiological parameters. In the present study,we investigated the effect of aquaculture site and translocation on different stocks of Mediterranean mussel, Mytilus galloprovincialis Lamarck, 1819. We used shell morphological ratios (width/length, height/length and height/width) and allometric relationships (volume-length and shell weight-length) in addition to condition index (wet meat weight/total weight) and mortality to compare cultivated and translocated mussels from the northern Adriatic Sea, Croatia. Mussels were collected from five cultivation areas (Lim, Pomer, Raša, Vabriga and Budava), and then moved for three months to experimental station at marine protected area in Lim Bay. Differences between cultivated mussels suggested morphological plasticity, although translocation weakly affected allometric relationships. Decrease in condition index was pronounced in translocated mussels, indicating adaptation to different local environmental conditions. High post translocation mortality was observed in mussels transferred to the experimental area. Our study shows plastic response of M. galloprovincialis, which can provide a useful information of mussel stocks selection in aquaculture.

Physiological Indices of Maricultured Mussel Mytilus galloprovincialis Lamarck, 1819 in Istria, Croatia: Seasonal and Transplantation Effect

AbstractTransplantation of mussels to new aquaculture sites is currently being undertaken in Istria (Croatia) to increase production, minimize potential seasonal urban contamination, and secure safer seafood products. The purpose of this study was to determine the effects of season and transplantation on physiological indices (condition index and meat yield) of mussels, Mytilus galloprovincialis. Mussels were obtained from five aquaculture areas in Istria (central Lim Bay, Raša, Budava, Vabriga, and Pomer) and moved to a reference site in inner Lim Bay from April to July. Results showed that both season and transplantation affected physiological indices of mussels. Higher physiological indices were found in cultivated mussels from all areas in summer compared to spring, except in mussels from Vabriga and Pomer. In addition, higher physiological indices were found in mussels transplanted from central Lim Bay to the reference site in inner Lim Bay. Transplantation negatively affected physiological indices of mussels from Vabriga and Pomer, with no change in mussels from Raša and Budava. In conclusion, mussels from all areas exhibit satisfactory commercial results (≥15%) during the peak summer harvesting season. For most transplanted mussels, transplantation itself represents additional stress, bringing into focus adaptation processes, stock genetics, and favorable ecological conditions.

Mytilus galloprovincialis (Lamarck, 1819) as host of Mytilicola orientalis (Mori, 1935) in the northern Adriatic Sea: presence and effect

The transfer of mariculture bivalves into new areas is increasing the risk of spreading Mytilicola copepods in native bivalve populations. Mytilicola orientalis was spread from Japan to native oysters and mussels in the Mediterranean. However, the presence of M. orientalis and its effect on the Mediterranean mussel, Mytilus galloprovincialis, are so far under-investigated. Our study shows the first identification of M. orientalis in mussels from the northern Adriatic Sea. M. orientalis was detected in mussels, from the Lim Bay, around the mariculture area of M. galloprovincialis. The adult stages of the copepod occur in mussels from May to August. The mean intensity of infestation and prevalence reached the maximum in August and was positively related to seawater temperature and salinity. We found one to three parasites per host individual, and most of the infested mussels were infested by one single parasite. Using the condition index, we found a relation between mussel physiology and seasonal cycle. Number of parasites was not related to the applied condition index. Our study established no difference in M. galloprovincialis condition index between infested and uninfested individuals, suggesting that the presence of M. orientalis does not adversely affect the mussel physiology to a measurable extent. However, our study reports the first record in the Adriatic Sea for M. orientalis.

137Cs distribution in the northern Adriatic Sea

Anthropogenic radioactivity in the northern Adriatic Sea was assessed by analyzing 137Cs activity concentrations in seawater, sediment, and marine organisms in the period from 2006 to 2010. The investigated locations were the Po River delta, Lim Bay and the Rovinj coastal area. At all locations 137Cs activity concentrations in seawater were lower than pre-Chernobyl values (<4 Bq m−3). The highest 137Cs activity concentrations in sediment were found in the Po River delta (8.70 Bq kg−1). The data indicate that some species, such as sardine Sardina pilchardus, red mullet Mullus barbatus and the brown alga Fucusvirsoides are better bioaccumulators of 137Cs than others. In addition, the ERICA Assessment Tool was used to calculate the total dose rates for reference organisms included in the software, as well as for specific sampled marine organisms.

Element differences and evaluation of the dietary intake from farmed oysters and mussels collected at different sites along the Croatian coast of the Adriatic Sea

Concentrations of the macroelements Ca, Mg, K and Na, microelements Cr, Cu, Fe, Mn and Zn and toxic metals As, Cd, Hg and Pb were determined in oysters and mussels collected at farming sites in the northern, central and south Adriatic Sea. Mean concentrations in oysters were (mgkg−1): As 4.51, Ca 1551, Cd 1.44, Cu 53.6, Cr 0.23, Fe 45.9, Hg 0.047, K 2476, Mg 895, Mn 2.80, Na 7148, Pb 0.52, Zn 675. Mean values in mussels were (mgkg−1): As 4.71, Ca 601, Cd 0.37, Cu 1.91, Cr 0.29, Fe 49.6, Hg 0.026, K 2246, Mg 1043, Mn 2.49, Na 7992, Pb 0.58, Zn 28.8. Significant differences in the concentrations of toxic metals and elements were found in oysters and mussels among farming sites. Obtained Cd, Hg and Pb concentration in oysters and mussels were lower than the limit levels set by the European Community. However, Cd concentration exceeded the limit level of 1mgkg−1 in oysters from all locations except Lim Bay. The estimated daily intakes (EDIs) indicate that oysters are a good source of Ca, Cu and Zn. The higher Cu and Zn concentrations than the toxicity reference values suggest a risk for consumers if larger quantities of oysters are consumed frequently.

Ecology and taxonomy of potentially toxic Pseudo-nitzschia species in Lim Bay (north-eastern Adriatic Sea)

The population dynamics of Pseudo-nitzschia in relation to environmental factors was investigated from March 2002 to July 2008 in Lim Bay, in the north-eastern Adriatic Sea. Domoic acid was monitored in the breeding population of Mytilus galloprovincialis from 2005 to 2008. The principal-component analysis of environmental parameters showed that the system is mostly temperature driven. The phytoplankton community was mainly composed of diatoms. Pseudo-nitzschia was the dominant diatom, present in 60% of samples, with a maximum (1.6 × 10 6 cells L −1) contribution up to 97% of the total diatom abundance. Morphological analysis revealed Pseudo-nitzschia manii and potentially toxic Pseudo-nitzschia pseudodelicatissima, Pseudo-nitzschia pungens, Pseudo-nitzschia fraudulenta and Pseudo-nitzschia calliantha as the dominant species in blooms. Pseudo-nitzschia abundance positively correlated to temperature, phosphate and ammonia in accordance with its maximal abundance in the summer/autumn period when fish farms had a maximum impact on the environment. Domoic acid was detected in M. galloprovincialis in concentrations below regulatory limits, ranging from 0.097 to 0.8721 μg g −1 in five cases from April to October 2005 in Lim Bay, but so far it is not clear which of the species was responsible for DA production. This study is also the first record of P. manii, P pungens and P. fraudulenta species in the Adriatic Sea.

Lipophilic Toxin Profile in Mytilus galloprovincialis during Episodes of Diarrhetic Shellfish Poisoning (DSP) in the N.E. Adriatic Sea in 2006

Dinophysis spp. blooms and related shellfish toxicity events of diarrhetic shellfish poisoning (DSP) have been the most reported toxicity event through the Croatian National monitoring program. With the aim to characterize the DSP toxin profile in shellfish farmed in Croatia, for the first time a complete analysis of the toxin profile of Croatian mussels has been carried out using the LC-MS/MS technique. The obtained results showed okadaic acid (OA) as the main toxin contaminating Croatian mussels at that time. The maximum concentration of OA in shellfish tissue was recorded 12 days after the Dinophysis fortii bloom, thus suggesting that rapid growth of the toxin level in the shellfish occurred in the first week after the bloom while it was slower in the second week. Furthermore, the presence of only OA at concentrations which could endanger human health suggests D. fortii as the main organism responsible for the toxic event that occurred in Lim Bay. The presence of gymnodimine and spirolides in Croatian mussel has been detected for the first time, while the presence of yessotoxin and pectenotoxin-2 is confirmed.

Is Yessotoxin the Main Phycotoxin in Croatian Waters?

With the aim of investigating whether yessotoxin (YTX) is responsible for diarrhetic shellfish poisoning (DSP) events in Croatian waters, three different methods were combined: a modified mouse bioassay (MBA) that discriminates YTX from other DSP toxins, the enzyme-linked immunosorbent assay method (ELISA) and liquid chromatography-mass spectrometry (LC-MS/MS). Among 453 samples of mussels and seawater analyzed in 2007, 10 samples were DSP positive. Results obtained by the modified MBA method revealed that most of the samples were positive for YTX, with the exception of samples from Lim Bay (LB 1) The ELISA method also identified the presence of YTX in these samples. DSP toxin profiles showed the presence of okadaic acid (OA) in three, and YTX in four out of nine samples that were analyzed by LC-MS/MS. The phytoplankton community structure pattern revealed Lingulodinium polyedrum (Stein) Dodge, which was present in the water prior to and/or during toxicity events at low concentrations (80 to 1440 cells L−1), as a potential YTX producing species. It is proposed that L. polyedrum cells accumulated in mussels and the subsequently observed toxicity may be related to metabolism after ingestion, resulting in carboxy YTX as the major analog in the mussel.