Is the Illegal Trade of Glass Eels (Anguilla anguilla) Increasing the Spread of Disease? A Case of EVEX.

The European eel (Anguilla anguilla) is a catadromous species that inhabits the rivers of the Adriatic watershed in Croatia. It is a critically endangered fish species, according to the IUCN, due to its declining abundance in European rivers caused by overfishing and trafficking and by diseases caused by nematodes, pathogenic bacteria and viruses. An illegal parcel of glass eels was confiscated at the Zagreb Airport and was intended to re-populate Croatian rivers. Barcoding was employed to determine species affiliation, and a thorough health check was carried out. This study reports the evaluation of gross lesions, histological findings, and EVEX virus isolation and identification. Since the confiscated glass eels were of unknown origin and given the serological and genetic similarities of EVA and EVEX, we designed primers and probes for almost whole genome sequencing to elucidate the origin of glass eels based on viral phylogeny. Bayesian phylogeny showed that the isolated strain had the most common ancestor with a Danish isolate and likely evolved from the French isolate of EVEX. These findings are discussed in light of the divergence of recently isolated strains and their possible contribution to the decrease of the abundance of the European eel in European waters.

A plant-based diet supplemented with Hermetia illucens alone or in combination with poultry by-product meal: one step closer to sustainable aquafeeds for European seabass.

BACKGROUND: Increasing demand for high-value fish species and pressure on forage fish is challenging aquaculture to ensure sustainable growth by replacing protein sources in aquafeeds with plant and terrestrial animal proteins, without compromising the economic value and quality of the final fish product. In the present study, the effects of a plant protein-based diet (CV), two plant-based diets in which graded amounts of plan protein mixtures were replaced with Hermetia illucens meal alone (VH10) or in combination with poultry by-product meal (PBM) (VH10P30), a fishmeal (FM) diet (CF) and an FM diet supplemented with H. illucens (FH10) on growth performance, gut health and homeostasis of farmed subadult European seabass were tested and compared. RESULTS: Fish fed the VH10 and VH10P30 diets showed the highest specific growth rates and lowest feed conversion ratios among the tested groups. Expectedly, the best preservation of PI morphology was observed in fish fed the CF or FH10 diets, while fish fed the CV diet exhibited significant degenerative changes in the proximal and distal intestines. However, PBM supplementation mitigated these effects and significantly improved all gut morphometric parameters in the VH10P30 group. Partial substitution of the plant mixture with insect meal alone or PBM also induced most BBM genes and activated BBM enzymes, suggesting a beneficial effect on intestinal digestive/absorption functions. Regarding intestinal microbiota, fish fed diets containing H. illucens meal (FH10, VH10, VH10P30) had the highest richness of bacterial communities and abundance of beneficial genera such as Lactobacillus and Bacillus. On the other hand, fish fed CV had the highest microbial diversity but lost a significant component of fish intestinal microbiota, the phylum Bacteroidetes. Finally, skin pigmentation most similar to that of farmed or even wild seabass was also observed in the fish groups fed CF, FH10 or VH10P30. CONCLUSION: Plant-based diets supplemented with PBM and H. illucens pupae meal have great potential as alternative diets for European seabass, without affecting growth performance, gut homeostasis, or overall fitness. This also highlights the importance of animal proteins in diets of European seabass, as the addition of a small amount of these alternative animal protein sources significantly improved all measured parameters.

Plankton diversity in Anthropocene: Shipping vs. aquaculture along the eastern Adriatic coast assessed through DNA metabarcoding.

Coastal ecosystems globally are exposed to the most pervasive anthropogenic activities, caused by a suite of human infrastructure and enterprises such as shipping ports, aquaculture facilities, fishing, and tourism. These anthropogenic activities may lead to changes in ecosystem biodiversity, followed by loss of ecosystem functioning and services. Shipping industry and aquaculture have also been recognized as the main vectors for introduction of marine non-indigenous species (NIS) worldwide. In this study, we used DNA metabarcoding-based methods to investigate plankton biodiversity under varying anthropogenic pressures (shipping and bivalve aquaculture) along the eastern Adriatic coast (the northernmost part of the Mediterranean Sea). Our comparative assessment revealed similar community structures among investigated coastal locations (Northern, Central and Southern Adriatic). When the whole plankton communities were considered, they did not differ significantly between port and aquaculture sites. However, the proportion of the unique zOTUs in the port samples was remarkably higher than that in aquaculture sites (40.5% vs 8.2%), indicating that port areas may receive higher abundance and species richness of NIS than aquaculture sites. Further important difference between the two types of anthropogenically impacted habitats was a high abundance of three notorious invaders - M. leidyi, M. gigas, and H. elegans in late summer at the aquaculture site in Northern Adriatic. Therefore, the plankton community of the area is under pressure not only from aquaculture activities, but also establishment of NIS. Port areas are probably under greater introduction pressure from NIS, but aquaculture sites may experience greater community changes due to their establishment.

Population Genetic Structure and Connectivity of the European Lobster Homarus gammarus in the Adriatic and Mediterranean Seas.

Highly selective fishing has the potential to permanently change the characteristics within a population and could drive the decline of genetic diversity. European lobster is an intensively fished crustacean species in the Adriatic Sea which reaches high market value. Since knowledge of population structure and dynamics is important for effective fisheries management, in this study, we used 14 neutral microsatellites loci and partial mitochondrial COI region sequencing to explore population connectivity and genetic structure by comparing samples from the Adriatic Sea and the adjacent basins of the Mediterranean Sea. The obtained results suggest that neutral genetic diversity has not been significantly affected by decrease in population size due to overfishing, habitat degradation and other anthropogenic activities. Global genetic differentiation across all populations was low (F ST = 0.0062). Populations from the Adriatic Sea were panmictic, while genetic differentiation was found among populations from different Mediterranean basins. Observed gene flow for European lobster suggest that populations in the north eastern Adriatic act as a source for surrounding areas, emphasizing the need to protect these populations by establishing interconnected MPAs that will be beneficial for both fisheries and conservation management.

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.

Spatial connectivity pattern of expanding gilthead seabream populations and its interactions with aquaculture sites: a combined population genetic and physical modelling approach.

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

Evidence of subtle genetic structure in the sympatric species Mullus barbatus and Mullus surmuletus (Linnaeus, 1758) in the Mediterranean Sea.

Using thirteen microsatellite loci for Mullus barbatus and Mullus surmuletus collected in the Mediterranean Sea, the biogeographic boundaries, genetic distribution among and within basins and the impact of prolonged exploitation in both species were investigated as a basis for understanding their population dynamics and for improving Mullus spp. stock management. Different level of diversity indices among these co-occurring species were obtained, with M. barbatus showing higher allele richness and higher mean observed and expected heterozygosity than M. surmuletus. Reduced contemporary effective population size (Ne) and M-ratio values found in both species likely reflects recent demographic changes, due to a combination of high fishing pressures, habitat fragmentation and naturally occurring fluctuations in population size. Different patterns of genetic connectivity among populations sampled within the Mediterranean were observed for both species. Higher genetic structure was found for M. barbatus as opposed to a more homogenous pattern observed in M. surmuletus samples. Adriatic populations, previously considered panmictic and isolated from other Mediterranean regions, showed geographical partitioning within the basin but also population connectivity with the northern Ionian and Tyrrhenian Seas. Our results highlight the need for temporal sampling in understanding the complex pattern of population connectivity in the Mediterranean, particularly for management purposes.

Spillover of the Atlantic bluefin tuna offspring from cages in the Adriatic Sea: A multidisciplinary approach and assessment.

During routine monitoring of commercial purse seine catches in 2011, 87 fingerling specimens of scombrids were collected in the southern Adriatic Sea. Sequencing of the mitochondrial DNA control region locus inferred that specimens belonged to the Atlantic bluefin tuna, Thunnus thynnus (Linnaeus, 1758) (N = 29), bullet tuna, Auxis rochei (Risso, 1810) (N = 30) and little tunny, Euthynnus alletteratus, Rafinesque, 1810 (N = 28). According to previously published growth parameters, the age of the collected specimens was estimated at approximately 30-40 days, suggesting they might have been spawned in the Adriatic Sea, contrary to the current knowledge. A coupled modelling system with hydrodynamic (ROMS) and individual based model (IBM-Ichthyop) was set up to determine the location of the spawning event. Numerical simulations with the IBM model, both backward and forward in time, indicate commercial tuna cages in the middle Adriatic coastal area as possible spawning location. The two other non-commercial species likely opportunistically use the positive environmental (abiotic and biotic) conditions to spawn in the same area.

Ecological role of bluefin tuna (Thunnus thynnus) fish farms for associated wild fish assemblages in the Mediterranean Sea.

The ecological effects of tuna fish farms are largely undocumented. This study confirmed their high capacity to attract surrounding wild fish. The aggregation effect persisted year round, without detectable seasonal differences. Farm impact was restricted to close proximity of the sea cages, and was more prominent over the bottom than in the water column strata. Tuna fish farms proved to be high energy trophic resources, as indicated by the enhanced fitness status of two focal species, bogue and seabream. Under abundant food supply, seabream appear to allocate the majority of energy reserves to gonad development. Farm associated bogue had greater parasite loads, with no detrimental effect on fitness status. Overall, tuna fish farms can be regarded as population sources for aggregated wild fish, and under the no fishing conditions within the leasehold areas, can serve as functional marine protected areas.

Combined Use of Morphological and Molecular Tools to Resolve Species Mis-Identifications in the Bivalvia The Case of Glycymeris glycymeris and G. pilosa.

Morphological and molecular tools were combined to resolve the misidentification between Glycymeris glycymeris and Glycymeris pilosa from Atlantic and Mediterranean populations. The ambiguous literature on the taxonomic status of these species requires this confirmation as a baseline to studies on their ecology and sclerochronology. We used classical and landmark-based morphometric approaches and performed bivariate and multivariate analyses to test for shell character interactions at the individual and population level. Both approaches generated complementary information. The former showed the shell width to length ratio and the valve asymmetry to be the main discriminant characters between Atlantic and Mediterranean populations. Additionally, the external microsculpture of additional and finer secondary ribs in G. glycymeris discriminates it from G. pilosa. Likewise, landmark-based geometric morphometrics revealed a stronger opisthogyrate beak and prosodetic ligament in G. pilosa than G. glycymeris. Our Bayesian and maximum likelihood phylogenetic analyses based on COI and ITS2 genes identified that G. glycymeris and G. pilosa form two separate monophyletic clades with mean interspecific divergence of 11% and 0.9% for COI and ITS2, respectively. The congruent patterns of morphometric analysis together with mitochondrial and nuclear phylogenetic reconstructions indicated the separation of the two coexisting species. The intraspecific divergence occurred during the Eocene and accelerated during the late Pliocene and Pleistocene. Glycymeris pilosa showed a high level of genetic diversity, appearing as a more robust species whose tolerance of environmental conditions allowed its expansion throughout the Mediterranean.

Morphological plasticity and phylogeny in a monogenean parasite transferring between wild and reared fish populations.

It is widely accepted that disease interactions between cultured and wild fish occur repeatedly, although reported cases have mainly relied just on the observation of similar symptoms in affected populations. Whether there is an explicit pathogen transfer between fish stocks, or each develops its own pathogen population, has been insufficiently studied and rarely supported by molecular tools. In this study, we used population dynamics and genetic structure of the monogenean Furnestinia echeneis in reared and neighbouring wild sea bream to indicate pathogen transfer, characterized by the phenotypic plasticity of the parasite attachment apparatus and the lack of phylogenetic differentiation. The observed pattern of genetic variation inferred by nuclear DNA Internal Transcribed Spacer 1 (ITS1) and mtDNA cytochrome C oxidase 1 (COI), between parasite populations is most likely caused by a recent shared demographic history like a reduced species area in the last glacial period. In spite of such recent expansion that populations underwent, F. echeneis shows differentiation in haptor morphometry as an adaptive trait in closely related populations at the aquaculture site. This suggests that differentiation in morphology may occur relatively rapidly in this species and that adaptive forces, not the speciation process, drives this monogenean parasitation. On the other hand, the observed phylogenetic inertia suggests a low to moderate gene flow (based on F ST ) between parasites in cultured and wild fish, evidencing for the first time the transfer of pathogens at the aquaculture site inferred by a molecular tool.