Željka Trumbić

Expression analysis of the Atlantic bluefin tuna (Thunnus thynnus) pro-inflammatory cytokines, IL-1β, TNFα1 and TNFα2 in response to parasites Pseudocycnus appendiculatus (Copepoda) and Didymosulcus katsuwonicola (Digenea)

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

Reproductive cycle and gonad development of Venus verrucosa L. (Bivalvia: Veneridae) in Kaštela Bay, Adriatic Sea

Abstract The warty venus (Venus verrucosa Linnaeus, 1758) is commercially one of the most important bivalves in the Mediterranean Sea. Studies of reproductive cycle and gonad development of this species were conducted in Kaštela Bay, eastern Adriatic Sea, in a period between March 2009 and February 2010. We analysed sex ratios, gonad developmental stages, perimeter of the oocytes, and length at sexual maturity using standard histological techniques. Sex ratios did not significantly deviate from the proportion of 1 : 1 (χ2=0.228, p=0.632) and hermaphroditic animals were not found. Venus verrucosa showed continuous annual gametogenic activity, without a resting period. Mean values of oocyte perimeter ranged from 163.7±72.4 µm in March to 370.57±95.16 µm in October. Significant correlation was found between mean gonad indices and temperature. Changes in condition index approximately followed changes in the reproductive cycle, while observed decrease in condition index in the period from July to November reflected prolongation of the spawning period. Although the reproductive cycle of V. verrucosa has been studied in other parts of the Mediterranean previously, this study provides the first qualitative data on maturation stages for this species in the eastern Adriatic as well as quantitative data on oocyte perimeters, that were not included in previous investigations.

First Report of Cryptosporidium sp. (Coccidia, Apicomplexa) Oocysts in the Black Mussel (Mytilus galloprovincialis) Reared in the Mali Ston Bay, Adriatic Sea

ABSTRACT Cryptosporidium spp. are obligate intracellular apicomplexan parasites that infect epithelial cells of the gastrointestinal systems of a wide range of vertebrate hosts, including humans. Its importance as a serious public health threat was recognized only since the HIV pandemic. Because of the particular filter feeding behavior of bivalves, these marine organisms are susceptible to the accumulation of Cryptosporidium spp. oocysts from the environment and their retention for a certain time, acting as potential zoonotic reservoirs. To preliminary evaluate the presence of Cryptosporidium spp. oocysts in cultured bivalves from the Mali Ston Bay, Adriatic Sea, we have analyzed individuals from a black mussel (Mytilus galloprovincialis) population by immunofluorescence, over a one year period at four different locations. Overall one-year prevalence of the Cryptosporidium spp. was 16.8%, and was correlated with the presumptive number of E. coli in the shellfish and seawater and abiotic factors (temperature, salinity, oxygen), suggesting the necessity for the updating of existing sanitary control measures in Croatia.

Anisakis simplex complex: ecological significance of recombinant genotypes in an allopatric area of the Adriatic Sea inferred by genome-derived simple sequence repeats☆

The genus Anisakis includes nine species which, due to close morphological resemblance even in the adult stage, have previously caused many issues in their correct identification. Recently observed interspecific hybridisation in sympatric areas of two closely related species, Anisakis simplex sensu stricto (s.s.) and Anisakis pegreffii, has raised concerns whether a F1 hybrid generation is capable of overriding the breeding barrier, potentially giving rise to more resistant/pathogenic strains infecting humans. To assess the ecological significance of anisakid genotypes in the Adriatic Sea, an allopatric area for the two above-mentioned species, we analysed data from PCR-RFLP genotyping of the ITS region and the sequence of the cytochrome oxidase 2 (cox2) mtDNA locus to discern the parental genotype and maternal haplotype of the individuals. Furthermore, using in silico genome-wide screening of the A. simplex database for polymorphic simple sequence repeats or microsatellites in non-coding regions, we randomly selected potentially informative loci that were tested and optimised for multiplex PCR. The first panel of microsatellites developed for Anisakis was shown to be highly polymorphic, sensitive and amplified in both A. simplex s.s. and A. pegreffii. It was used to inspect genetic differentiation of individuals showing mito-nuclear mosaicism which is characteristic for both species. The observed low level of intergroup heterozygosity suggests that existing mosaicism is likely a retention of an ancestral polymorphism rather than a recent recombination event. This is also supported by allopatry of pure A. simplex s.s. and A. pegreffii in the geographical area under study.

Genetic characterization of wild and farmed European seabass in the Adriatic sea: assessment of farmed escapees using a Bayesian approach

&NA; Hybridizations between domesticated escaped farmed fish and wild conspecifics may increase genetic diversity or break down locally adapted gene complexes, thus reducing competitive ability and overall fitness. We examined the genetic structure of six farmed populations of European seabass, Dicentrarchus labrax, of different geographical origin, and ten neighbouring wild populations in the Adriatic Sea using 10 microsatellite loci. All loci were polymorphic, with mean expected heterozygosity >74% in all 16 populations. Overall number of alleles as well as short‐term effective population size were smaller in farmed (A = 18.7; Ne = 56.1) than in wild populations (A = 21.2; Ne = 180.6). The global FST of 0.0672 across loci showed significant population subdivision. Strong genetic differences between farmed fish and their wild conspecifics enabled the identification of seabass escapees back to their origins and the estimation of the extent of hybridization. Bayesian assignment analyses clustered wild populations together, whereas each farmed populations was assigned to a separate cluster. Intraspecific hybridization was highest in wild populations in areas impacted by fish farms, where highly admixed populations had decreased genetic diversity. The results of this study represent a solid foundation required to establish a genetic register of European seabass wild and farmed populations in the eastern Adriatic Sea that are required to establish a seabass DNA register.

Autochthonous Bacterial Isolates Successfully Stimulate In vitro Peripheral Blood Leukocytes of the European Sea Bass (Dicentrarchus labrax)

Commercially available probiotics are routinely administered as feed supplements in aquaculture important species. Among them, the European sea bass (Dicentrarchus labrax) is the most widely reared fish in the Mediterranean, whose rearing systems are highly variable between countries, affecting at some level the sustainability of production. After random isolation of autochthonous gut bacteria of the sea bass, their identification and pathogenicity testing, we have selected three potentially probiotic isolates; Pseudoalteromonas sp., Alteromonas sp., and Enterovibrio coralii. Selected isolates were tested and their immunostimulative efficiency was compared with a commercially available Lactobacillus casei isolate, inferring inflammatory, apoptotic and anti-pathogen response of sea bass’ peripheral blood leukocytes. 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 to evaluate early kinetics of the responses. Best immunostimulative properties were observed in Pseudoalteromonas-stimulated leukocytes, followed by Alteromonas sp. and L. casei, while Enterovibrio coralii failed to induce significant stimulation. Based on such in vitro assay intestinal autochthonous bacterial isolates showed to have better immunostimulative effect in sea bass compared to aquaculture-widely used L. casei, and further steps need to engage tank and field feeding trials to evaluate long-term prophylactic suitability of the chosen isolates. A panel of biomarkers that represent pro-/anti-inflammatory, pro-/anti-apoptotic, and anti-bacteria/viral responses of the fish should be taken into consideration when evaluating the usefulness of the potential probiotic in aquaculture.

Molecular and Cellular Response to Experimental Anisakis pegreffii (Nematoda, Anisakidae) Third-Stage Larval Infection in Rats

Background: Anisakiasis is a zoonotic disease caused by accidental ingestion of live Anisakis spp. third-stage larvae present in raw or undercooked seafood. Symptoms of this emerging infectious disease include mild-to-severe abdominal pain, nausea, and diarrhea. Some patients experience significant allergic reactions. Aims: In order to better understand the onset of anisakiasis, we aimed to: (i) histopathologically describe severe inflammatory/hemorrhagic infection site lesions in Sprague-Dawley rats experimentally infected with Anisakis pegreffii larvae; and (ii) qualitatively and quantitatively characterize the transcriptomes of affected tissues using RNA-Seq. Methodology: The experiment was performed on 35 male rats, sacrificed at 5 time points (6, 10, 18, 24, and 32 h post-infection). Gastric intubation was performed with 10 A. pegreffii larvae (N = 5 infected rats per time point) or 1.5 ml of saline (external control N = 2 rats). 16 pools, seven for muscle tissues and nine for stomach tissues, were created to obtain robust samples for estimation of gene expression changes depicting common signatures of affected versus unaffected tissues. Illumina NextSeq 500 was used for paired-end sequencing, while edgeR was used for count data and differential expression analyses. Results: In total, there were 1372 (855 up and 517 down) differentially expressed (DE) genes in the Anisakis-infected rat stomach tissues, and 1633 (1230 up and 403 down) DE genes in the muscle tissues. Elicited strong local proinflammatory reaction seems to favor the activation of the interleukin 17 signaling pathway and the development of the T helper 17-type response. The number of DE ribosomal genes in the Anisakis-infected stomach tissue suggests that A. pegreffii larvae might induce ribosomal stress in the early infection stage. However, the downstream pathways and post-infection responses require further study. Histopathology revealed severe inflammatory/hemorrhagic lesions caused by Anisakis infection in the rat stomach and muscle tissues in the first 32 h. The lesion sites showed infiltration by polymorphonuclear leukocytes (predominantly neutrophils and occasional eosinophils), and to a lesser extent, macrophages. Conclusion: Understanding the cellular and molecular mechanisms underlying host responses to Anisakis infection is important to elucidate many aspects of the onset of anisakiasis, a disease of growing public health concern.